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An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes
Accepted Manuscript Title: eAn efficient ribitol-specific dehydrogenase from Enterobacter aerogenes Author: Ranjitha Singh Raushan Singh In-Won Kim Sujan Sigdel Vipin C. Kalia Yun Chan Kang Jung-Kul Lee PII: DOI: Reference:
S0141-0229(15)00030-7 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.02.004 EMT 8730
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
8-9-2014 6-2-2015 11-2-2015
Please cite this article as: Singh R, Singh R, Kim I-W, Sigdel S, Kalia VC, Kang YC, Lee J-K, An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes, Enzyme and Microbial Technology (2015), http://dx.doi.org/10.1016/j.enzmictec.2015.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Running title: Ribitol dehydrogenase from Enterobacter aerogenes
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An efficient ribitol-specific dehydrogenase from
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Enterobacter aerogenes
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Ranjitha Singh1, Raushan Singh1, In-Won Kim1, Sujan Sigdel1, Vipin C. Kalia2, Yun Chan
Department of Chemical Engineering, Konkuk University,
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Kang3*, Jung-Kul Lee1*
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1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, South Korea Microbial Biotechnology and Genomics, CSIR-Institute of Genomics and Integrative
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Biology, Delhi University Campus, Mall Road, Delhi-110007, India
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Department of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, South Korea
*Author for correspondence. E-mail: [email protected], Tel: +82-2-450-3505; Fax: +82-2-458-0879 E-mail: [email protected], Tel: +82-2-3290-3268; Fax: +82-2-928-3584
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Highlights
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> It reports a highly active ribitol dehydrogenase (EaRDH) from Enterobacter
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aerogenes. > Among various polyols, EaRDH exhibits activity only towards ribitol.
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> EaRDH shows the highest catalytic efficiency among all characterized RDHs. > Docking
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analyses shed light on the molecular basis of its unusually high activity.
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Abstract
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An NAD+-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH)
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was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was
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amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel
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affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C,
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respectively. Among various polyols, EaRDH exhibited activity only towards ribitol, with Km,
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Vmax, and kcat/Km values of 10.3 mM, 185 U mg-1, and 30.9 s-1 mM-1, respectively. The enzyme
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showed strong preference for NAD+ and displayed no detectable activity with NADP+.
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Homology modeling and sequence analysis of EaRDH, along with its biochemical properties,
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confirmed that EaRDH belongs to the family of NAD+-dependent ribitol dehydrogenases, a
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member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest
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activity and unique substrate specificity among all known RDHs. Homology modeling and
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docking analysis shed light on the molecular basis of its unusually high activity and substrate
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specificity.
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Key words: short-chain dehydrogenase/reductase, Enterobacter aerogenes, ribitol
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dehydrogenase, homology modeling
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1. Introduction
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Rare sugars can be applied in the food and pharmaceutical industries to stimulate our immune
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system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks
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for anticancer and antiviral drugs (e.g., L-nucleosides). Due to these multiple applications, rare
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sugars are considered to be of great interest to researchers. Ribulose, in the D- or L-form, is
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scarce in nature and is therefore classified as a rare sugar. D-ribulose is useful in pharmaceutical
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chemistry as a starting material for the synthesis of branched pentoses [1]. Additionally, a
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derivative of D-ribulose is already found in some artificial sweeteners (e.g., sucroribulose).
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Furthermore, D-ribulose is an important precursor for the synthesis of oligosaccharides, amino
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sugars, and glycosides [2]. Microbial and enzymatic reactions are very suitable for converting D-
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sugars to various L-sugars [3]. D-ribulose can be successfully converted to L-ribulose using
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microbial and enzymatic reactions. The rationale for research on L-ribulose production has
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mainly been the possibility of isomerizing it to L-ribose which is useful as a building block for
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the manufacture of glycoconjugates, oligonucleotides, L-aptamers, and antiviral or anticancer L-
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nucleosides, as the starting material for the production of L-allose and L-altrose, and in potential
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therapeutics against HBV and Epstein-Barr virus [4]. Consequently, ribulose is of great industrial
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interest in light of its broad applications.
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Ribitol dehydrogenase (ribitol: NAD+ 2-oxidoreductase; RDH; EC 1.1.1.56) catalyzes the
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conversion of ribitol (also called adonitol) to D-ribulose [5, 6]. Ribitol is a naturally occurring
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polyol, which is commercially available and affordable (178 USD per 100 g) [3, 7]. Ribitol
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dehydrogenase (RDH) belongs to the family of short chain dehydrogenases/ reductases (SDR,
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also known as the short-chain oxidoreductase/SCOR family). SDRs constitute one of the largest
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enzyme superfamilies, with presently over 160,000 members in sequence databases and over 300
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crystal structures deposited in the PDB [8, 9]. The large SDR superfamily is of ancient origin,
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with most members being equidistantly related at the 20–30% residue identity level. The
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members display a highly similar α/β folding pattern with a Rossmann fold, consisting of a
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central β-sheet flanked by α-helices, typical of and common to other oxidoreductases [10]. Most
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SDR enzymes have a core structure of 250–350 residues in length, frequently with N- or C-
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terminal transmembrane domains or signal peptides, or form parts of multi-enzyme complexes
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[11].
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Although many short-chain dehydrogenases from different microorganisms have been
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identified, purified, and biochemically characterized, the search for new dehydrogenases with
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better biochemical properties for industrial applications, such as rare sugar production and in
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pharmaceutical therapies, continues. The quest for more effective RDHs, with emphasis on a
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broad pH activity profile, high selectivity, stability, and efficient catalysis, resulted in our
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identification of the ribitol dehydrogenase (EaRDH) from Enterobacter aerogenes KCTC 2190.
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The gene from Enterobacter aerogenes was chosen over other bacterial species because bacteria
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of Enterobacter sp. are known for their unique oxidative metabolism. In the present paper, we
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report the cloning, heterologous expression, and characterization of a new ribitol dehydrogenase
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(EaRDH) from Enterobacter aerogenes KCTC 2190. Based on its characteristics and sequence,
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EaRDH has been classified as a member of the SDR superfamily. In the present investigation,
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we provide experimental evidence that EaRDH is an NAD+-dependent oxidoreductase that
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exclusively utilizes ribitol as the sole substrate among various polyols, thus exhibiting very high
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substrate specificity towards ribitol. Furthermore, in order to understand the molecular details of
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substrate recognition and catalysis, a structural study using homology modeling and docking of
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EaRDH was performed with ribitol as the substrate. The results from this study should guide
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future mutagenesis studies to investigate the role of individual amino acids in the catalytic
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mechanism of RDHs.
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2. Materials and Methods
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2.1. Materials
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Reagents for polymerase chain reaction (PCR), Ex-Taq DNA polymerase and T4 DNA ligase,
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were purchased from Takara (Takara Corp., Shiga, Japan). The genomic DNA extraction kit and
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the pGEM-T easy vector were purchased from Promega (Madison, WI, USA). Restriction
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enzymes were obtained from New England Biolabs (Ipswich, MA, USA). The pET 28(a)
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expression vector, plasmid isolation kit, and Ni-NTA super flow column for purification were
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from Novagen (Madison, WI, USA), Bioneer Co. (Daejeon, Korea), and Qiagen (Hilden,
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Germany) respectively. Oligonucleotide primers were obtained from Macrogen Inc. (Seoul,
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South Korea). Electrophoresis reagents were from Bio-Rad (Hercules, CA, USA) and all
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chemicals for assay were from Sigma-Aldrich (St. Louis, MO, USA).
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2.2. Bacterial strains and culture conditions
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Enterobacter aerogenes KCTC 2190 was obtained from the Korean Collection for Type Culture
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(KCTC, Daejeon, South Korea). The strain was grown aerobically at 30°C in nutrient broth
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containing beef extract 3 g l-1 and peptone 5 g l-1 with constant shaking (200 rpm). E. coli DH5α
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and E. coli BL21-CodonPlus (DE3)-RIL were used as hosts for plasmid transformation and
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expression, respectively. Both the E. coli strains were cultivated in Luria-Bertani (LB) medium
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at 37°C and 200 rpm on a rotary shaker with the addition of 50 μg ml-1 kanamycin and/or 50 μg
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ml-1 chloramphenicol to select for the presence of plasmids when appropriate.
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2.3. PCR amplification of rdh gene from E. aerogenes KCTC 2190
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The E. aerogenes genomic sequence and RDH protein sequence were accessed from the National
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Center for Biotechnology Information (www.ncbi.nlm.nih.gov). E. aerogenes was grown in
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culture, and the genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit
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(Promega). The rdh gene was amplified using polymerase chain reaction (PCR) from E.
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aerogenes genomic DNA using the 2 oligonucleotide primers 5–
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AGGATCCATGAATACTTCTCTTAGCG–3 (BamHI restriction site is underlined) and 5–
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CTCGAGTAAATCAACGCTGTTAGGC–3 (XhoI restriction site is underlined).
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2.4. Cloning and expression of rdh gene from E. aerogenes KCTC 2190
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The amplified rdh gene with BamHI and XhoI restriction sites was first cloned into the pGEM-T
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easy vector and transformed into E. coli DH5α. The cloned rdh gene was confirmed to be free of
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any point mutation by DNA sequencing performed at the Biotechnology Center of the Macrogen
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Sequencing System (Macrogen Inc. Seoul, South Korea). The rdh gene was then subcloned into
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the BamHI-XhoI sites of pET28a. The resultant pET28a–rdh, under the control of the T7
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promoter, expressed a ribitol dehydrogenase with a hexa-histidine (6His) tag at the N–terminus
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of the protein. The recombinant plasmid (pET28a–rdh) was then transformed into E. coli BL21-
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CodonPlus (DE3)-RIL. The recombinant protein was expressed using 0.1 mM isopropyl-β- D-
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thiogalactopyranoside (IPTG) at 25°C for ~6 hours with shaking at 200 rpm. The induced cells
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were harvested by centrifugation at 4°C for 15 min at 4,000 × g, rinsed with lysis buffer (50 mM
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NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and stored at −20°C.
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2.5. Purification of recombinant EaRDH from E. aerogenes KCTC 2190
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To purify the recombinant 6His-tagged ribitol dehydrogenase (RDH), cell pellets were
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resuspended in lysis buffer at 2–5 ml/g wet weight. The cell suspension was incubated on ice for
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30 min in the presence of 1 mg ml-1 lysozyme. Cell disruption was carried out by sonication at
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4°C for 5 min and the lysate was centrifuged at 14,000 × g for 20 min at 4°C to remove the cell
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debris. The cell-free extract was applied onto a Ni-NTA Super flow column (3.4 × 13.5 cm,
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Qiagen) previously equilibrated using lysis buffer. Unbound proteins were washed out from the
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column using the wash buffer (50 mM NaH2PO4, 300 mM NaCl, 90 mM imidazole, pH 8.0). The
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recombinant enzyme was eluted from the column using the elution buffer (50 mM NaH2PO4, 300
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mM NaCl, 250 mM imidazole, pH 8.0).
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2.6. Protein quantification and determination of molecular mass
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Protein concentrations were determined by the Bradford method using bovine serum albumin as
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the standard protein [12]. The molecular mass of the native enzyme was determined by gel
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filtration chromatography following the previously described procedure [13]. The subunit
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molecular weight was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis
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(SDS-PAGE) under denaturing conditions, using the pre-stained ladder marker (Bio-Rad) as
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reference proteins [14, 15].
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2.7. Enzyme assay
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The activity of EaRDH was determined spectrophotometrically by monitoring the increase in
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absorbance at 340 nm, A340 (εNADH = 6.22 mM−1 cm−1) at 25°C. The RDH assay mixture for
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oxidation consisted of 2 mM NAD+, 200 mM ribitol, and an enzyme solution in 100 mM Tris-
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Glycine-NaOH (pH 11.0). The reaction was started by the addition of the substrate (ribitol). One
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unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol NADH
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min−1 under the assay conditions. Enzyme assays were conducted in triplicate using freshly
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purified EaRDH enzyme.
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2.8. Optimum pH and temperature
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The pH optimum of the purified enzyme was determined using the following 3 buffer systems:
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100 mM sodium acetate (pH 4.0–6.0), 100 mM Tris-HCl (pH 6.0–9.0), and 100 mM Tris-
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glycine-NaOH (pH 8.0–12.0). The pH values were measured at 25°C in solutions similar to the
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final reaction mixtures, with the exception that the enzyme and NAD+ were omitted. The optimal
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temperature for the activity of EaRDH was determined by assaying the enzyme samples over the
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range of 25–60 °C. The maximum activity was considered as 100%, and used as the reference in
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determining relative activities at different pH and temperature.
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Stability of the enzyme towards temperature was investigated by incubating the enzyme
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in 100 mM Tris-glycine-NaOH (pH 11.0) containing 5 mM Ca2+ at different temperatures. At
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certain time intervals, samples were withdrawn and residual activity was measured under
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standard assay conditions.
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2.9. Substrate and coenzyme specificity
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Specificity of the recombinant EaRDH was determined by assaying the enzyme at optimum pH
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at 25°C with different substrates ribitol (adonitol), xylitol, mannitol, galactitol (dulcitol), sorbitol
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(glucitol), meso-erythritol, myo-inositol, glycerol, allitol, and D/L-threitol and D/L-arabinitol
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(arabitol) (200 mM). The coenzyme specificity of EaRDH was also determined using NAD+ and
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NADP+ as coenzymes. The maximum activity was considered as 100%, and relative activities for
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other substrates were determined.
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2.10. Effect of metal ions and ICP-MS analysis
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The effect of metal ion on the activity of the purified EaRDH was determined following the
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previously described procedure [13]. The presence and amount of metals (Mg2+, Mn2+, Zn2+,
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Ba2+, Sn2+, Ni2+, and Ca2+) in the recombinant EaRDH protein was determined using inductively
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coupled plasma mass spectrometry (ICP-MS) following the previously described procedure [16].
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2.11. Determination of kinetic parameters
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Kinetic parameters of EaRDH were determined in 100 mM Tris-glycine-NaOH (pH 11.0) buffer,
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5 mM Ca2+, 0.25–300 mM substrate, and 0.05–2 mM coenzyme. The kinetic constants were
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obtained from at least triplicate measurements of the initial rates at varying concentrations of
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ribitol and NAD+. The kinetic parameters Km and Vmax, were determined from non-linear
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regression fitting of the Michaelis-Menten equation using Prism 5 (Graphpad Software, Inc., CA,
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USA). The data represent the average of all statistically relevant data with a standard deviation of
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less than 10%.
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2.12. Homology modeling and sequence analysis
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Utilizing the X-ray crystal structures of clavulanic acid dehydrogenase (CAD) from
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Streptomyces clavuligerus and the galactitol dehydrogenase (GatDH) from Rhodobacter
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sphaeroides D as the templates, a three-dimensional (3D) multiple template homology model
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was generated using the Build homology models (MODELER) [17] module in Discovery Studio
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3.1 (DS3.1, Accelrys Software Inc., San Diego, CA, USA). EaRDH had the highest sequence
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identities of 34.5% and 26.1% with S. clavuligerus CAD (PDB code: 2JAH; resolution 1.80 Å)
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[18] and R. sphaeroides D GatDH (PDB code: 2WSB; resolution 1.25 Å) [19], respectively. The
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3D homology modeling and sequence analysis of EaRDH and Zymomonas mobilis RDH
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(ZmRDH) were done as described previously [20, 21]. ZmRDH , a broad substrate specific
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enzyme, was used as a representative model to compare substrate binding sites of EaRDH and
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ZmRDH.
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2.13. Cofactor and substrate docking
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The validated EaRDH and ZmRDH 3D models were used for docking and post-docking analysis.
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Hydrogen atoms were first added to the 3D models, and then the added hydrogen atoms were
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minimized for stable energy conformation and relaxation of the conformations from close
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contacts under the CHARMm forcefield [22]. Iterative rounds of docking were used to identify
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the optimum substrate-docked conformation. The cofactor (NAD+) and substrate (ribitol)
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molecules were first energy-minimized under the same forcefield and docked into the cofactor
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and substrate binding pockets of EaRDH and ZmRDH using CDOCKER, a MD simulation-
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annealing-based algorithm module from DS 3.1 [23]. First, NAD+ was docked into the
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coenzyme-binding domain of the generated models. Then, the energy-minimized complex of the
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enzyme and NAD+ was used as the receptor molecule for docking with its natural substrate
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ribitol. Before docking, the structure of the proteins, substrate, and their complexes were
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subjected to energy minimization using the CHARMm force field implemented in DS 3.1. Then,
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a full potential final minimization was used to refine substrate (ligand) orientation. The docked
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conformation of the substrate with the lowest energy was retrieved from CDOCKER for post-
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docking analysis.
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3. Results and Discussion
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3.1. Identification of a ribitol dehydrogenase (RDH) gene from E. aerogenes KCTC 2190
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Utilizing the whole-genome sequence (REFSEQ: NC_015663.1) of E. aerogenes KCTC 2190,
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an rdh gene encoding an unnamed protein product (REFSEQ:YP_004594905.1) was identified.
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The unnamed protein possessed the NAD-binding glycine-rich Rossmann fold domain ([T–G–
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X3–G–X–G]) (position 13-20; where X is any amino acid) and the active site residues (Asn112,
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Ser140, Tyr153 and Lys157) highly conserved among SDRs (Fig. 1). Furthermore, the deduced
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protein product had a sequence identity of ~25% (~41% similarity) with Zymomonas mobilis
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RDH [24], the only recombinant RDH reported so far. Members of the SDR protein family are
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known to share low overall sequence identity of 15 to 30% despite the common fold that is
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highly conserved [9]. Thus, based on these bioinformatics, the gene from E. aerogenes was
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annotated as a putative rdh, suggesting that it might encode an RDH. The putative rdh gene from
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E. aerogenes, with a total length of 729 base pairs (bp), encodes a polypeptide of 242 amino
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acids, with a calculated molecular mass of 25.8 kDa.
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3.2. Heterologous expression and purification of EaRDH
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A dehydrogenase assay carried out with extracts of E. coli BL21-CodonPlus (DE3)-RIL
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harboring pET 28(a)–EaRDH revealed the presence of a high level of RDH activity compared
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with the control E. coli BL21-CodonPlus (DE3)-RIL cells harboring empty circular plasmid pET
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28(a). The recombinant EaRDH enzyme was purified to electrophoretic homogeneity from the
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culture supernatant by Ni-affinity column chromatography.
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3.3. Determination of molecular mass and quaternary structure
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The subunit molecular mass of the enzyme was 25.8 kDa, as determined by PAGE in the
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presence of SDS (Fig. 2A). Gel filtration chromatography on a Sephacryl S–300 high-resolution
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column resulted in the elution of EaRDH as a symmetrical peak between aldolase and
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conalbumin, corresponding to a molecular weight of approximately 110 kDa (Fig. 2B). These
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results indicate that the enzyme migrates as a tetramer in gel filtration chromatography and exists
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as an active tetramer in solution as well, which is typical of other RDHs [6, 25, 26].
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3.4. Optimum pH and temperature
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The effect of pH on enzyme activity is shown in Fig. 3A. Maximal activity was achieved under
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high alkaline condition at pH 11.0 (pH above 10.0), consistent with the notion that an ionized
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Tyr153 (pKa = 10) is involved in catalysis. Similar pH dependence has been observed for other
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members of the sugar alcohol dehydrogenase group [27]. The optimal temperature for the
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dehydrogenation reaction was found to be 45°C (Fig. 3B). The enzyme retained more than 90%
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of the maximum activity when assayed at 50°C, with more than 85% retained at 55°C, and more
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than 80% at 60°C (Fig. 3B). Supplementary Fig. S1 shows the percentage of residual activity vs.
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incubation time, which followed first-order decay with half-lives of 25.7, 15.3, 7.5, 3.1, and 0.45
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h at 40°C, 45°C, 50°C, 55°C, and 60°C, respectively.
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3.5. Substrate and coenzyme specificity
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The characterization of EaRDH as an RDH allowed the investigation of its substrate specificity
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for various polyols. EaRDH was specific for ribitol and did not show any activity towards other
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polyols. To the best of our knowledge, this is the first report of a RDH showing ribitol-limited
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specificity, unlike other RDHs characterized previously exhibiting broad substrate specificities
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(see Table 1). A measurement with ribitol as the substrate showed that EaRDH was exclusively a
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NAD+-dependent enzyme showing essentially no activity with NADP+.
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3.6. Effects of metal ions and ICP-MS analysis
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The EaRDH enzyme was purified followed by extensive dialysis in the presence of 10 mM
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EDTA. As shown in Table 2, we found that the enzyme activity was mostly lost (~97%) after
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EDTA treatment. This indicates that EaRDH is a metal-dependent enzyme. The activity of the
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EaRDH enzyme was completely inhibited by Hg2+ and partially activated by Fe3+. Almost
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complete recovery of enzyme activity was observed in the presence of 1 mM K+ (96%) and Cu2+
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(90%). EaRDH activity was significantly stimulated by Ba2+ or Mg2+. However, the highest level
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of activation was achieved using Ca2+ (5 mM). Ca2+ enhanced the activity about 2.45-fold. The
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enhancement in activity of EaRDH was found to be dependent on the presence of divalent metal
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ions in the following order: Ca2+ >> Ba2+ >> Mg2+ >> Mn2+≈ Co2+.
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To investigate the presence of divalent metal ions in purified EaRDH and assess the ability of EaRDH to bind metal ions, the recombinant protein was subjected to ICP-MS analysis.
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Based on the apparent molecular mass of 25.8 kDa, 1 μmol of enzyme contained 0.50 ± 0.04
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μmol of Mg2+. The ICP-MS data provided support for 1 metal binding site per dimer, as
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observed in other SDRs such as dTDP-6-deoxy- L-lyxo-4-hexulose reductase [28], xylitol
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dehydrogenase [29], and galactitol dehydrogenase [19]. The concentrations of Mn2+, Zn2+, Ba2+,
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Ni2+, and Sn2+ were below the detection limit, and no appreciable amount of Ca2+ was detected.
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Mg2+ enzymes have often been substituted with Ca2+/Cd2+ because of their similar electronic
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properties [30]. In addition, the coordination chemistry of Ca2+ is closely related to Mg2+. At
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coordination number (CN) = 6, the ionic radii of Ca2+, Cd2+, and Mg2+ are 1.00, 0.95, and 0.72 A,
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respectively, whereas at CN = 8 they are 1.12, 1.10, and 0.89 A, respectively [31]. Without a
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detailed study of metal binding in EaRDH through crystallography or NMR studies, it is not
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possible to explain why Ca2+ is better than Mg2+ at restoring EaRDH activity.
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3.7. Kinetic parameters of EaRDH
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Initial velocities were determined in the standard assay mixture at pH 11.0. Ribitol had
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hyperbolic saturation curves and the corresponding double-reciprocal plots were linear (data not
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shown). Fig. 4 shows kinetic parameters for EaRDH activity with increasing ribitol
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concentrations from 0.25 to 300 mM. Maximum enzyme activity was obtained with a ribitol
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concentration of about 200 mM under the given experimental conditions. The Km values of
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EaRDH for ribitol and NAD+ were found to be 10.3 and 0.16 mM, respectively (Table 1). The
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Vmax and kcat/Km values were 185 U mg-1 and 30.9 s-1 mM-1, respectively. The observed activity
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of EaRDH (185 U mg-1) was the highest of any reported RDH to date (Table 1). The detailed
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kinetic studies for only two previously characterized RDHs from Klebsiella aerogenes evolved
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strain A and Z. mobilis have been reported so far. The kcat/Km,Ribitol value (30.9 s-1 mM-1 ) for
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EaRDH was the highest compared with the kcat/Km,Ribitol of 28.7 s-1 mM-1 and 0.41 s-1 mM-1 for
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KaRDH [32] and ZmRDH [24], respectively. Although a number of sequences for characterized
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and putative RDHs are present in the NCBI database, detailed quantitative kinetic studies are
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lacking.
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3.8. Homology modeling of EaRDH
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The overall secondary structure of the modeled EaRDH enzyme was similar to that of CAD from
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S. clavuligerus and GatDH from R. sphaeroides D (Supplementary Fig. S2). The generated
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EaRDH model was validated using the validation tool PROCHECK [33] in order to confirm the
308
consistency of the model. The Profile-3D score of the model was 93.21 against a maximum
309
expected score of 106.72, which compares well to the scores of 109.47 and 112.5 for the
310
coordinates from 2JAH and 2WSB. The built model was also evaluated by superimposing it onto
311
the template crystal structures and the obtained RMSD values were 0.44 Å and 0.6 Å with
312
respect to 2JAH and 2WSB, based on the Cα atoms (Supplementary Fig. S3). The structural
313
model of EaRDH obtained by molecular modeling consists of a classical α/β Rossmann fold
314
pattern commonly found in the SDR family [10, 34]. Upon superimposition, the coenzyme-
315
binding motif and the active site residues of EaRDH superimposed well upon the coenzyme-
316
binding motif and the catalytic residues of S. clavuligerus CAD and R. sphaeroides D GatDH,
317
respectively (Supplementary Fig. S4). Despite low sequence homology among SDR family
318
members, motifs that define classical SDRs are apparent in EaRDH. Apart from the nucleotide-
319
binding motif and the active tetrad of Asn112–Ser140–Tyr153–Lys157, the EaRDH enzyme
320
possesses the conserved but slightly modified Ala87–Asn88–Ala89–Gly90 motif (sequence
321
important for the stabilization of the central β-sheet), a singular Asp60 (residue responsible for
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322
the stabilization of adenine ring pocket), and a Pro183–Gly184 motif (residues determining
323
reaction direction), followed by a conserved Thr188 (residue responsible for the H-bonding to
324
carboxamide of nicotinamide ring) [10, 11].
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3.9. Cofactor and substrate docking
327
In order to investigate the molecular basis responsible for the unique substrate specificity, we
328
constructed a structural model of EaRDH and docked the cofactor (NAD+) and the substrate
329
(ribitol) into the active site to mimic the interactions between the enzyme, cofactor, and substrate
330
(Supplementary Fig. S5). The axis of the NAD+ ran roughly parallel to the β-sheet and
331
perpendicular to the 6 Rossmann fold helices. The NAD+ was anchored to the enzyme with the
332
nicotinamide ring in the syn conformation, which is stabilized in this conformation by an
333
intramolecular hydrogen bond between the nicotinamide and the pyrophosphate. The coenzyme-
334
binding site is formed by residues 13–20, 38–40, 60–64, 88–90, and 188 (residues numbered
335
with respect to EaRDH). The nicotinamide ribose group is in a cleft surrounded by the conserved
336
catalytic residues Asn112, Tyr153, and Lys157. The loop formed by residues 13–20 is the well-
337
known glycine-rich motif that characterizes many dehydrogenases because its composition and
338
conformational flexibility facilitate interaction with the coenzyme. Near the carboxyl-terminal
339
edge of the dinucleotide fold, bounded on one side by the si-face of nicotinamide, a rather
340
narrow groove serves as the binding site of the substrate (Fig. 5). Because of this narrow width
341
of the groove, the substrate must slip in and bind with its backbone nearly parallel to the
342
nicotinamide plane such that the C2 carbon of the substrate is positioned directly below the C4
343
carbon of the cofactor (Fig. 5A). Upon substrate docking, the C2 hydroxyl of ribitol, which is
344
oxidized by the enzyme to a carbonyl, hydrogen bonds with the catalytic base Tyr153 of
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17 Page 17 of 35
EaRDH. Ribitol, due to its stereochemical arrangement, could slide into this narrow groove,
346
providing all the favorable interactions to ensure the oxidation of ribitol at C2 (Fig. 5B). The C2
347
carbon, which transfers its hydride to NAD+ during the course of the reaction, has a distance of
348
3.6 Å to the nicotinamide C4, suitable for hydride transfer. An attempt to dock other polyol
349
substrates into the narrow substrate-binding pocket of EaRDH resulted in collisions between
350
molecules or a lack of feasible hydrogen bonding between the protein and the cofactor.
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We have modeled a structure of ZmRDH, a broad-specificity RDH, in order to compare
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351
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substrate binding sites of EaRDH and ZmRDH. ZmRDH is the only characterized RDH with
353
known sequence available in NCBI. A total of 28 amino acid residues including the active-site
354
residues were located within the 4.5Å radius of the substrate binding pocket (SBP) of both
355
ZmRDH and EaRDH. Upon alignment, the substrate binding sites of ZmRDH and EaRDH
356
shared only 21.4% of sequence identity (Supplementary Fig. S6), mostly due to the active site
357
residues of Asn112–Ser140–Tyr153–Lys157 (numbered with respect to EaRDH). Without
358
thorough determination of the role of each SBP residue of EaRDH, it is difficult to suggest the
359
molecular determinants of the narrow substrate specificity of EaRDH. The substrate binding site
360
cavities of EaRDH and ZmRDH exhibited volumes of 190Å3 and 256Å3, respectively. The shape
361
and volume of the cavities likely affect the substrate selectivity, and this, in turn, reflects
362
differences in the structure of EaRDH and ZmRDH as evident from the 3D structures (Fig. 6A
363
and B). The binding site cavity of ZmRDH is larger in the vicinity of the catalytic site residues
364
(Asn131–Ser157–Tyr170–Lys174) as compared to the smaller cavity volume of EaRDH. The
365
larger binding site cavity of ZmRDH can accommodate various substrate molecules because of
366
its large size, whereas the smaller EaRDH binding site cavity is not likely to allow the entry of
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18 Page 18 of 35
367
various polyol substrates. These findings indicate that the selection of substrate by the narrow
368
substrate binding pocket of EaRDH is more restricted than in previously reported ZmRDH.
369
4. Conclusion
371
In conclusion, we have cloned, expressed, and characterized a highly active RDH from E.
372
aerogenes. The purified recombinant protein showed RDH activity, confirming the identity of
373
the protein. The bio-physiochemical properties and homology modeling studies strongly affirm
374
that EaRDH is a member of the SDR family. The characterized RDH shares common
375
biochemical properties with previously known RDHs, but differs in enzymatic kinetics and
376
substrate specificity. EaRDH showed the highest turnover rate and catalytic efficiency among all
377
previously characterized RDHs. A detailed interpretation of the marked preference of EaRDH
378
for ribitol as its sole substrate and its high activity will rely on 3D structural analysis, which is
379
our future direction of study.
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Acknowledgements
382
This work was supported by the Energy Efficiency & Resources Core Technology Program of
383
the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial
384
resource from the Ministry of Trade, Industry & Energy, Republic of Korea (201320200000420).
385
This research was also supported by Basic Science Research Program through the National
386
Research Foundation of Korea funded by the Ministry of Education, Science and Technology
387
(NRF-2013R1A1A2007561). This work was supported by 2014 KU Brain Pool fellowship of
388
Konkuk University.
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19 Page 19 of 35
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References
390
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[3] Ahmed Z. Production of natural and rare pentoses using microorganisms and their enzymes.
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amino acid sequence of ribitol dehydrogenase-F, a mutant enzyme with improved xylitol
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dehydrogenase activity. J Protein Chem. 1999;18:489-95.
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[6] Adachi O, Fujii Y, Ano Y, Moonmangmee D, Toyama H, Shinagawa E, et al. Membrane-
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[7] Wymer N, & Taylor, P., inventors; Zuchem, Inc. assignee. Production of L-ribose and other rare sugars. United States patent US8642297. 2014 Feb 4. [8] Persson B, Kallberg Y. Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs). Chemico-Biological Interactions. 2013;202:111-5.
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[9] Kallberg Y, Oppermann U, Persson B. Classification of the short-chain
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dehydrogenase/reductase superfamily using hidden Markov models. FEBS J.
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[12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of
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protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. [13] Singh RK, Tiwari MK, Kim D, Kang YC, Ramachandran P, Lee JK. Molecular cloning and
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characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in
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enzymatic pretreatment of biomass. Appl Microbiol Biotechnol. 2012.
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[14] Lee KM, Kalyani D, Tiwari MK, Kim TS, Dhiman SS, Lee JK, et al. Enhanced enzymatic hydrolysis of rice straw by removal of phenolic compounds using a novel laccase from yeast
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Yarrowia lipolytica. Bioresour Technol. 2012;123:636-45.
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[15] Jagtap SS, Dhiman SS, Kim TS, Kim IW, Lee JK. Characterization of a novel endo-beta-
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1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis. Appl
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Microbiol Biotechnol. 2014;98:661-9.
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[16] Tiwari MK, Singh RK, Singh R, Jeya M, Zhao H, Lee JK. Role of conserved glycine in
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zinc-dependent medium chain dehydrogenase/reductase superfamily. J Biol Chem.
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2012;287:19429-39.
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[17] Sali A, Potterton L, Yuan F, van Vlijmen H, Karplus M. Evaluation of comparative protein modeling by MODELLER. Proteins. 1995;23:318-26. [18] MacKenzie AK, Kershaw NJ, Hernandez H, Robinson CV, Schofield CJ, Andersson I. Clavulanic acid dehydrogenase: structural and biochemical analysis of the final step in the
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biosynthesis of the beta-lactamase inhibitor clavulanic acid. Biochemistry. 2007;46:1523-33.
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[19] Carius Y, Christian H, Faust A, Zander U, Klink BU, Kornberger P, et al. Structural insight
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into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase
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from Rhodobacter sphaeroides D. J Biol Chem. 2010;285:20006-14.
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[20] Tiwari M, Lee JK. Molecular modeling studies of L-arabinitol 4-dehydrogenase of
an
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Hypocrea jecorina: its binding interactions with substrate and cofactor. J Mol Graph Model.
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2010;28:707-13.
[21] Moon HJ, Tiwari MK, Singh R, Kang YC, Lee JK. Molecular determinants of the cofactor
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specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase. Appl Environ
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Microbiol. 2012;78:3079-86.
te
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[22] Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M.
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CHARMM: A program for macromolecular energy, minimization, and dynamics
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calculations. Journal of Computational Chemistry. 1983;4:187-217.
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[23] Wu G, Robertson DH, Brooks CL, 3rd, Vieth M. Detailed analysis of grid-based molecular
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docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm. J Comput
451
Chem. 2003;24:1549-62.
452 453
[24] Moon HJ, Tiwari M, Jeya M, Lee JK. Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis. Appl Microbiol Biotechnol. 2010;87:205-14.
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[25] Muniruzzaman S, Kunihisa Y, Ichiraku K, Izumori K. Purification and characterization of a
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ribitol dehydrogenase from Enterobacter agglomerans strain 221e. Journal of Fermentation
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and Bioengineering. 1995;79:496-8.
460 461
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Purification and subunit structure. Biochem J. 1974;141:693-700.
[27] Lee D, Redfern O, Orengo C. Predicting protein function from sequence and structure. Nat
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[26] Taylor SS, Rigby PW, Hartley BS. Ribitol dehydrogenase from Klebsiella aerogenes.
Rev Mol Cell Biol. 2007;8:995-1005.
us
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[28] Blankenfeldt W, Kerr ID, Giraud MF, McMiken HJ, Leonard G, Whitfield C, et al. Variation on a theme of SDR. dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD) shows a
463
new Mg2+-dependent dimerization mode. Structure. 2002;10:773-86.
467 468
M
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466
dehydrogenase cosubstrate specificity. Structure. 2006;14:567-75. [30] Dudev T, Lim C. Principles governing Mg, Ca, and Zn binding and selectivity in proteins.
te
465
[29] Ehrensberger AH, Elling RA, Wilson DK. Structure-guided engineering of xylitol
Chem Rev. 2003;103:773-88.
[31] Shannon RD. Revised effective ionic radii and systematic studies of interatomic distances in
Ac ce p
464
an
462
469
halides and chalcogenides. Acta Crystallographica Section A: Crystal Physics, Diffraction,
470
Theoretical and General Crystallography. 1976;32:751-67.
471 472 473
[32] Burleigh BD, Rigby PW, Hartley BS. A comparison of wild-type and mutant ribitol dehydrogenases from Klebsiella aerogenes. Biochem J. 1974;143:341-52. [33] Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check
474
the stereochemical quality of protein structures. Journal of Applied Crystallography.
475
1993;26:283-91.
23 Page 23 of 35
476
[34] Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, et al. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem.
478
2002;277:25677-84.
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Optimum temperature
Metal ion
Specific activity (Units/mg)
Klebsiella aerogenes A
11.0
NR
Zn2+
NR
Rhodobacter sphaeroides Si4
10.0
45°C
NR
9.0–10.0
9.5–10.5
NR
Sn2+/ Na2+
NR
ce pt
Gluconobacter oxydans IFO 12528
50–60°C
ed
Enterobacter agglomerans
Km (mM)
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Optimum pH
59
144
Reference
Substrate (Ribitol)
0.66
10.4
Ribitola, xylitol, L-arabitol
[24]
0.08
6.3
Ribitol (100)b, xylitol (21), erythritol (12), D-sorbitol (10), D-arabitol (10)
[33]
32.2
Ribitol (100)c, allitol (66), L-arabitol (56.5), L-mannitol (44.5), xylitol (42.5), L-sorbitol (41), L-talitol (26.5), L-iditol (7), D-sorbitol (5), galactitol (3), erythritol (1)
[23]
1.2
Ribitol (100)d, xylitol (85.6), L-arabitol (79.8), D-arabitol (16.1), erythritol (6.8), D-sorbitol (3.4)
[6]
[22]
This study
2.36
29.2
Substrate specificity (relative % activity)
Coenzyme (NAD+)
M an
Organism
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Table 1. Biochemical and kinetic properties of ribitol dehydrogenases from various organisms
0.08
9.5
65°C
Mn2+
5.65
0.18
11.8
Enterobacter aerogenes KCTC 2190
11.0
45°C
Mg2+
185
0.16
10.3
Ribitol (100)f
Ac
Zymomonas mobilis
Ribitol (100)e, D-threitol (105), Lthreitol (98.7), L-arabitol (102), xylitol (91.2), glycerol (89.1), mannitol (84.9), galactitol (55.6), sorbitol (41.1)
NR, Not reported a Relative activity not specified b Values in parentheses indicate relative activity at 150 mM substrate concentration c Values in parentheses indicate relative activity at 10 mM substrate concentration d Values in parentheses indicate relative activity at 100 µM substrate concentration e Values in parentheses indicate relative activity at 100 mM substrate concentration f Values in parentheses indicate relative activity at 200 mM substrate concentration
25 Page 25 of 35
Table 2. Effects of different metal ions on the activity of EaRDH. The EDTA-treated enzyme was assayed in standard assay conditions with 1 mM or 5 mM metal ions. The activity of EaRDH before EDTA treatment was set as 100%. The metal-depleted apoenzyme retained
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only 3% activity relative to the native EaRDH enzyme. Each value represents the mean of
Mg2+
148 ± 4
Mn2+
161 ± 6
Zn2+
139 ± 3
Ca2+
193 ± 7
Fe3+
45.5 ± 4.8
27.2 ± 2.4
2+
90.1 ± 3.1
50.5 ± 6.1
119 ± 2
108 ± 4
0
0
142 ± 3
209 ± 6
95.7 ± 7.0
73.6 ± 3.5
Hg2+ Ba2+
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K+
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Co2+
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Cu
Relative activity (%) 5 mM 100 ± 4
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Before EDTA treatment
Relative activity (%) 1 mM 100 ± 2
Metal ions
479
cr
triplicate measurements and varied from the mean by not more than 15%.
162 ± 6 109 ± 8
61.9 ± 5.0 245 ± 8
26 Page 26 of 35
479
Figure legends
480
Fig. 1. Multiple sequence alignment of E. aerogenes RDH with selected short-chain
482
reductase/dehydrogenases (SDRs). EaRDH, Enterobacter aerogenes RDH (Accession number
483
YP_004594905.1); GoXDH, Gluconobacter oxydans xylitol dehydrogenase (Accession number
484
JC7939); RsGatDH, Rhodobacter sphaeroides galactitol dehydrogenase (Accession number
485
2WDZ); AbMDH, Agaricus bisporus mannitol dehydrogenase (Accession number 1H5Q);
486
CtHSD, Comamonas testosteroni 3β/17β-hydroxysteroid dehydrogenase (Accession number
487
1HXH); MmCR, Mus musculus carbonyl reductase (Accession number NP_031647.1);
488
BnACPR, Brassica napus enoyl-acyl carrier protein (acp) reductase (Accession number 1EDO);
489
BmGDH, Bacillus megaterium glucose dehydrogenase (Accession number P40288.1); KpBDH,
490
Klebsiella pneumonia meso-2,3-butanediol dehydrogenase (Accession number 1GEG); and
491
HsXR, Homo sapiens xylulose reductase (Accession number NP_057370.1). The four members
492
of the catalytic tetrad are indicated with red boxes. The glycine-rich consensus sequences that
493
have a structural role in coenzyme binding in all SDRs are indicated by the yellow box. Amino
494
acids conserved, semi-conserved, and weakly conserved are displayed in dark blue, blue, and
495
light blue, respectively.
cr
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496
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481
497
Fig. 2. Determination of the molecular mass of E. aerogenes RDH by SDS-PAGE and gel
498
filtration chromatography. (A) Overexpression and purification of recombinant EaRDH. Lane M,
499
molecular mass Marker; Lane 1, soluble fraction of E. coli (control) harboring empty pET 28a;
500
Lane 2, soluble fraction of E. coli harboring recombinant pET 28a–EaRDH; Lane 3, purified
501
recombinant EaRDH. (B) Determination of native molecular mass of EaRDH by gel filtration
27 Page 27 of 35
502
chromatography on a sephacryl S–300 high-resolution column (Amersham). The column was
503
calibrated with standard molecular mass proteins aldolase (158 kDa), conalbumin, (75 kDa),
504
bovine serum albumin (66 kDa), and ovalbumin (44 kDa).
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Fig. 3. Effects of pH (A) and temperature (B) on the activity of EaRDH. Enzyme assays were
507
carried out under standard conditions in the presence of 200 mM ribitol. Activities at the optimal
508
temperature and pH were defined as 100%. Each value represents the mean of triplicate
509
measurements and varied from the mean by not more than 10%. 100 mM sodium acetate buffer
510
(filled circles), 100 mM Tris-HCl buffer (open circle), and 100 mM Tris-glycine NaOH buffer
511
(inverted triangle).
an
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cr
506
M
512
Fig. 4. Effect of substrate concentration on the activity of EaRDH. RDH activity of the enzyme
514
was measured in the presence of the indicated concentration of substrate at pH 11.0. The data
515
represent an average of all statistically relevant data with a standard deviation of less than 15%.
te
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516
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513
517
Fig. 5. Molecular surface representation of the EaRDH model. (A) Stereo view and (B) front
518
view of the molecular surface representation of the substrate binding pocket of the EaRDH
519
model. The active site residues (Asn112, Ser140, Tyr153, and Lys157) of EaRDH are shown in
520
green carbon. Hydrogen bonds and π -π interactions are represented by dashed green and orange
521
lines, respectively. The path of hydride transfer between ribitol C2 and NAD+ C4 is marked with
522
green line. The hydrophobic region, hydrogen bond donor, and hydrogen bond acceptor atoms
523
are depicted in yellow, green, and brown, respectively. For clarity, only the nicotinamide ring of
524
NAD+ is presented.
28 Page 28 of 35
525
Fig. 6. Active site cavities of EaRDH and ZmRDH. The active site cavities were calculated using
527
DS 3.1 and rendered as mesh surfaces. Full views of the (A) EaRDH and (B) ZmRDH models.
528
Active site residues are colored with blue (ZmRDH) and green (EaRDH) colored carbon, and
529
other residues near the active site are colored with white (ZmRDH) and grey colored carbon
530
(EaRDH). NAD+ is shown as sticks with orange carbon and ribitol as ball and sticks with yellow
531
carbon.
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526
29 Page 29 of 35
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A
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B
Fig. 2 31 Page 31 of 35
M
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A
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B
Fig. 3 32 Page 32 of 35
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Fig. 4
33 Page 33 of 35
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B
d
M
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A
Fig. 5 34 Page 34 of 35
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Fig. 6 35 Page 35 of 35
S0141-0229(15)00030-7 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.02.004 EMT 8730
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
8-9-2014 6-2-2015 11-2-2015
Please cite this article as: Singh R, Singh R, Kim I-W, Sigdel S, Kalia VC, Kang YC, Lee J-K, An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes, Enzyme and Microbial Technology (2015), http://dx.doi.org/10.1016/j.enzmictec.2015.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Running title: Ribitol dehydrogenase from Enterobacter aerogenes
cr
An efficient ribitol-specific dehydrogenase from
us
Enterobacter aerogenes
an
Ranjitha Singh1, Raushan Singh1, In-Won Kim1, Sujan Sigdel1, Vipin C. Kalia2, Yun Chan
Department of Chemical Engineering, Konkuk University,
d
1
M
Kang3*, Jung-Kul Lee1*
2
te
1 Hwayang-Dong, Gwangjin-Gu, Seoul 143-701, South Korea Microbial Biotechnology and Genomics, CSIR-Institute of Genomics and Integrative
Ac ce p
Biology, Delhi University Campus, Mall Road, Delhi-110007, India
1
Department of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 136-713, South Korea
*Author for correspondence. E-mail: [email protected], Tel: +82-2-450-3505; Fax: +82-2-458-0879 E-mail: [email protected], Tel: +82-2-3290-3268; Fax: +82-2-928-3584
1 Page 1 of 35
Highlights
2
> It reports a highly active ribitol dehydrogenase (EaRDH) from Enterobacter
3
aerogenes. > Among various polyols, EaRDH exhibits activity only towards ribitol.
4
> EaRDH shows the highest catalytic efficiency among all characterized RDHs. > Docking
5
analyses shed light on the molecular basis of its unusually high activity.
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6
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7 8
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1
Abstract
an
9
An NAD+-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH)
11
was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was
12
amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel
13
affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C,
14
respectively. Among various polyols, EaRDH exhibited activity only towards ribitol, with Km,
15
Vmax, and kcat/Km values of 10.3 mM, 185 U mg-1, and 30.9 s-1 mM-1, respectively. The enzyme
16
showed strong preference for NAD+ and displayed no detectable activity with NADP+.
17
Homology modeling and sequence analysis of EaRDH, along with its biochemical properties,
18
confirmed that EaRDH belongs to the family of NAD+-dependent ribitol dehydrogenases, a
19
member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest
20
activity and unique substrate specificity among all known RDHs. Homology modeling and
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docking analysis shed light on the molecular basis of its unusually high activity and substrate
22
specificity.
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Key words: short-chain dehydrogenase/reductase, Enterobacter aerogenes, ribitol
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dehydrogenase, homology modeling
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1. Introduction
27
Rare sugars can be applied in the food and pharmaceutical industries to stimulate our immune
29
system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks
30
for anticancer and antiviral drugs (e.g., L-nucleosides). Due to these multiple applications, rare
31
sugars are considered to be of great interest to researchers. Ribulose, in the D- or L-form, is
32
scarce in nature and is therefore classified as a rare sugar. D-ribulose is useful in pharmaceutical
33
chemistry as a starting material for the synthesis of branched pentoses [1]. Additionally, a
34
derivative of D-ribulose is already found in some artificial sweeteners (e.g., sucroribulose).
35
Furthermore, D-ribulose is an important precursor for the synthesis of oligosaccharides, amino
36
sugars, and glycosides [2]. Microbial and enzymatic reactions are very suitable for converting D-
37
sugars to various L-sugars [3]. D-ribulose can be successfully converted to L-ribulose using
38
microbial and enzymatic reactions. The rationale for research on L-ribulose production has
39
mainly been the possibility of isomerizing it to L-ribose which is useful as a building block for
40
the manufacture of glycoconjugates, oligonucleotides, L-aptamers, and antiviral or anticancer L-
41
nucleosides, as the starting material for the production of L-allose and L-altrose, and in potential
42
therapeutics against HBV and Epstein-Barr virus [4]. Consequently, ribulose is of great industrial
43
interest in light of its broad applications.
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Ribitol dehydrogenase (ribitol: NAD+ 2-oxidoreductase; RDH; EC 1.1.1.56) catalyzes the
45
conversion of ribitol (also called adonitol) to D-ribulose [5, 6]. Ribitol is a naturally occurring
46
polyol, which is commercially available and affordable (178 USD per 100 g) [3, 7]. Ribitol
47
dehydrogenase (RDH) belongs to the family of short chain dehydrogenases/ reductases (SDR,
48
also known as the short-chain oxidoreductase/SCOR family). SDRs constitute one of the largest
4 Page 4 of 35
enzyme superfamilies, with presently over 160,000 members in sequence databases and over 300
50
crystal structures deposited in the PDB [8, 9]. The large SDR superfamily is of ancient origin,
51
with most members being equidistantly related at the 20–30% residue identity level. The
52
members display a highly similar α/β folding pattern with a Rossmann fold, consisting of a
53
central β-sheet flanked by α-helices, typical of and common to other oxidoreductases [10]. Most
54
SDR enzymes have a core structure of 250–350 residues in length, frequently with N- or C-
55
terminal transmembrane domains or signal peptides, or form parts of multi-enzyme complexes
56
[11].
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Although many short-chain dehydrogenases from different microorganisms have been
58
identified, purified, and biochemically characterized, the search for new dehydrogenases with
59
better biochemical properties for industrial applications, such as rare sugar production and in
60
pharmaceutical therapies, continues. The quest for more effective RDHs, with emphasis on a
61
broad pH activity profile, high selectivity, stability, and efficient catalysis, resulted in our
62
identification of the ribitol dehydrogenase (EaRDH) from Enterobacter aerogenes KCTC 2190.
63
The gene from Enterobacter aerogenes was chosen over other bacterial species because bacteria
64
of Enterobacter sp. are known for their unique oxidative metabolism. In the present paper, we
65
report the cloning, heterologous expression, and characterization of a new ribitol dehydrogenase
66
(EaRDH) from Enterobacter aerogenes KCTC 2190. Based on its characteristics and sequence,
67
EaRDH has been classified as a member of the SDR superfamily. In the present investigation,
68
we provide experimental evidence that EaRDH is an NAD+-dependent oxidoreductase that
69
exclusively utilizes ribitol as the sole substrate among various polyols, thus exhibiting very high
70
substrate specificity towards ribitol. Furthermore, in order to understand the molecular details of
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substrate recognition and catalysis, a structural study using homology modeling and docking of
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EaRDH was performed with ribitol as the substrate. The results from this study should guide
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future mutagenesis studies to investigate the role of individual amino acids in the catalytic
74
mechanism of RDHs.
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2. Materials and Methods
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2.1. Materials
79
Reagents for polymerase chain reaction (PCR), Ex-Taq DNA polymerase and T4 DNA ligase,
80
were purchased from Takara (Takara Corp., Shiga, Japan). The genomic DNA extraction kit and
81
the pGEM-T easy vector were purchased from Promega (Madison, WI, USA). Restriction
82
enzymes were obtained from New England Biolabs (Ipswich, MA, USA). The pET 28(a)
83
expression vector, plasmid isolation kit, and Ni-NTA super flow column for purification were
84
from Novagen (Madison, WI, USA), Bioneer Co. (Daejeon, Korea), and Qiagen (Hilden,
85
Germany) respectively. Oligonucleotide primers were obtained from Macrogen Inc. (Seoul,
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South Korea). Electrophoresis reagents were from Bio-Rad (Hercules, CA, USA) and all
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chemicals for assay were from Sigma-Aldrich (St. Louis, MO, USA).
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2.2. Bacterial strains and culture conditions
90
Enterobacter aerogenes KCTC 2190 was obtained from the Korean Collection for Type Culture
91
(KCTC, Daejeon, South Korea). The strain was grown aerobically at 30°C in nutrient broth
92
containing beef extract 3 g l-1 and peptone 5 g l-1 with constant shaking (200 rpm). E. coli DH5α
93
and E. coli BL21-CodonPlus (DE3)-RIL were used as hosts for plasmid transformation and
94
expression, respectively. Both the E. coli strains were cultivated in Luria-Bertani (LB) medium
6 Page 6 of 35
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at 37°C and 200 rpm on a rotary shaker with the addition of 50 μg ml-1 kanamycin and/or 50 μg
96
ml-1 chloramphenicol to select for the presence of plasmids when appropriate.
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2.3. PCR amplification of rdh gene from E. aerogenes KCTC 2190
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The E. aerogenes genomic sequence and RDH protein sequence were accessed from the National
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Center for Biotechnology Information (www.ncbi.nlm.nih.gov). E. aerogenes was grown in
101
culture, and the genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit
102
(Promega). The rdh gene was amplified using polymerase chain reaction (PCR) from E.
103
aerogenes genomic DNA using the 2 oligonucleotide primers 5–
104
AGGATCCATGAATACTTCTCTTAGCG–3 (BamHI restriction site is underlined) and 5–
105
CTCGAGTAAATCAACGCTGTTAGGC–3 (XhoI restriction site is underlined).
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2.4. Cloning and expression of rdh gene from E. aerogenes KCTC 2190
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The amplified rdh gene with BamHI and XhoI restriction sites was first cloned into the pGEM-T
109
easy vector and transformed into E. coli DH5α. The cloned rdh gene was confirmed to be free of
110
any point mutation by DNA sequencing performed at the Biotechnology Center of the Macrogen
111
Sequencing System (Macrogen Inc. Seoul, South Korea). The rdh gene was then subcloned into
112
the BamHI-XhoI sites of pET28a. The resultant pET28a–rdh, under the control of the T7
113
promoter, expressed a ribitol dehydrogenase with a hexa-histidine (6His) tag at the N–terminus
114
of the protein. The recombinant plasmid (pET28a–rdh) was then transformed into E. coli BL21-
115
CodonPlus (DE3)-RIL. The recombinant protein was expressed using 0.1 mM isopropyl-β- D-
116
thiogalactopyranoside (IPTG) at 25°C for ~6 hours with shaking at 200 rpm. The induced cells
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were harvested by centrifugation at 4°C for 15 min at 4,000 × g, rinsed with lysis buffer (50 mM
118
NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and stored at −20°C.
119
2.5. Purification of recombinant EaRDH from E. aerogenes KCTC 2190
121
To purify the recombinant 6His-tagged ribitol dehydrogenase (RDH), cell pellets were
122
resuspended in lysis buffer at 2–5 ml/g wet weight. The cell suspension was incubated on ice for
123
30 min in the presence of 1 mg ml-1 lysozyme. Cell disruption was carried out by sonication at
124
4°C for 5 min and the lysate was centrifuged at 14,000 × g for 20 min at 4°C to remove the cell
125
debris. The cell-free extract was applied onto a Ni-NTA Super flow column (3.4 × 13.5 cm,
126
Qiagen) previously equilibrated using lysis buffer. Unbound proteins were washed out from the
127
column using the wash buffer (50 mM NaH2PO4, 300 mM NaCl, 90 mM imidazole, pH 8.0). The
128
recombinant enzyme was eluted from the column using the elution buffer (50 mM NaH2PO4, 300
129
mM NaCl, 250 mM imidazole, pH 8.0).
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2.6. Protein quantification and determination of molecular mass
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Protein concentrations were determined by the Bradford method using bovine serum albumin as
133
the standard protein [12]. The molecular mass of the native enzyme was determined by gel
134
filtration chromatography following the previously described procedure [13]. The subunit
135
molecular weight was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis
136
(SDS-PAGE) under denaturing conditions, using the pre-stained ladder marker (Bio-Rad) as
137
reference proteins [14, 15].
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2.7. Enzyme assay
8 Page 8 of 35
The activity of EaRDH was determined spectrophotometrically by monitoring the increase in
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absorbance at 340 nm, A340 (εNADH = 6.22 mM−1 cm−1) at 25°C. The RDH assay mixture for
142
oxidation consisted of 2 mM NAD+, 200 mM ribitol, and an enzyme solution in 100 mM Tris-
143
Glycine-NaOH (pH 11.0). The reaction was started by the addition of the substrate (ribitol). One
144
unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol NADH
145
min−1 under the assay conditions. Enzyme assays were conducted in triplicate using freshly
146
purified EaRDH enzyme.
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2.8. Optimum pH and temperature
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The pH optimum of the purified enzyme was determined using the following 3 buffer systems:
150
100 mM sodium acetate (pH 4.0–6.0), 100 mM Tris-HCl (pH 6.0–9.0), and 100 mM Tris-
151
glycine-NaOH (pH 8.0–12.0). The pH values were measured at 25°C in solutions similar to the
152
final reaction mixtures, with the exception that the enzyme and NAD+ were omitted. The optimal
153
temperature for the activity of EaRDH was determined by assaying the enzyme samples over the
154
range of 25–60 °C. The maximum activity was considered as 100%, and used as the reference in
155
determining relative activities at different pH and temperature.
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Stability of the enzyme towards temperature was investigated by incubating the enzyme
157
in 100 mM Tris-glycine-NaOH (pH 11.0) containing 5 mM Ca2+ at different temperatures. At
158
certain time intervals, samples were withdrawn and residual activity was measured under
159
standard assay conditions.
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2.9. Substrate and coenzyme specificity
9 Page 9 of 35
Specificity of the recombinant EaRDH was determined by assaying the enzyme at optimum pH
163
at 25°C with different substrates ribitol (adonitol), xylitol, mannitol, galactitol (dulcitol), sorbitol
164
(glucitol), meso-erythritol, myo-inositol, glycerol, allitol, and D/L-threitol and D/L-arabinitol
165
(arabitol) (200 mM). The coenzyme specificity of EaRDH was also determined using NAD+ and
166
NADP+ as coenzymes. The maximum activity was considered as 100%, and relative activities for
167
other substrates were determined.
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2.10. Effect of metal ions and ICP-MS analysis
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The effect of metal ion on the activity of the purified EaRDH was determined following the
171
previously described procedure [13]. The presence and amount of metals (Mg2+, Mn2+, Zn2+,
172
Ba2+, Sn2+, Ni2+, and Ca2+) in the recombinant EaRDH protein was determined using inductively
173
coupled plasma mass spectrometry (ICP-MS) following the previously described procedure [16].
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2.11. Determination of kinetic parameters
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Kinetic parameters of EaRDH were determined in 100 mM Tris-glycine-NaOH (pH 11.0) buffer,
177
5 mM Ca2+, 0.25–300 mM substrate, and 0.05–2 mM coenzyme. The kinetic constants were
178
obtained from at least triplicate measurements of the initial rates at varying concentrations of
179
ribitol and NAD+. The kinetic parameters Km and Vmax, were determined from non-linear
180
regression fitting of the Michaelis-Menten equation using Prism 5 (Graphpad Software, Inc., CA,
181
USA). The data represent the average of all statistically relevant data with a standard deviation of
182
less than 10%.
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2.12. Homology modeling and sequence analysis
10 Page 10 of 35
Utilizing the X-ray crystal structures of clavulanic acid dehydrogenase (CAD) from
186
Streptomyces clavuligerus and the galactitol dehydrogenase (GatDH) from Rhodobacter
187
sphaeroides D as the templates, a three-dimensional (3D) multiple template homology model
188
was generated using the Build homology models (MODELER) [17] module in Discovery Studio
189
3.1 (DS3.1, Accelrys Software Inc., San Diego, CA, USA). EaRDH had the highest sequence
190
identities of 34.5% and 26.1% with S. clavuligerus CAD (PDB code: 2JAH; resolution 1.80 Å)
191
[18] and R. sphaeroides D GatDH (PDB code: 2WSB; resolution 1.25 Å) [19], respectively. The
192
3D homology modeling and sequence analysis of EaRDH and Zymomonas mobilis RDH
193
(ZmRDH) were done as described previously [20, 21]. ZmRDH , a broad substrate specific
194
enzyme, was used as a representative model to compare substrate binding sites of EaRDH and
195
ZmRDH.
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2.13. Cofactor and substrate docking
198
The validated EaRDH and ZmRDH 3D models were used for docking and post-docking analysis.
199
Hydrogen atoms were first added to the 3D models, and then the added hydrogen atoms were
200
minimized for stable energy conformation and relaxation of the conformations from close
201
contacts under the CHARMm forcefield [22]. Iterative rounds of docking were used to identify
202
the optimum substrate-docked conformation. The cofactor (NAD+) and substrate (ribitol)
203
molecules were first energy-minimized under the same forcefield and docked into the cofactor
204
and substrate binding pockets of EaRDH and ZmRDH using CDOCKER, a MD simulation-
205
annealing-based algorithm module from DS 3.1 [23]. First, NAD+ was docked into the
206
coenzyme-binding domain of the generated models. Then, the energy-minimized complex of the
207
enzyme and NAD+ was used as the receptor molecule for docking with its natural substrate
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11 Page 11 of 35
ribitol. Before docking, the structure of the proteins, substrate, and their complexes were
209
subjected to energy minimization using the CHARMm force field implemented in DS 3.1. Then,
210
a full potential final minimization was used to refine substrate (ligand) orientation. The docked
211
conformation of the substrate with the lowest energy was retrieved from CDOCKER for post-
212
docking analysis.
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3. Results and Discussion
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3.1. Identification of a ribitol dehydrogenase (RDH) gene from E. aerogenes KCTC 2190
217
Utilizing the whole-genome sequence (REFSEQ: NC_015663.1) of E. aerogenes KCTC 2190,
218
an rdh gene encoding an unnamed protein product (REFSEQ:YP_004594905.1) was identified.
219
The unnamed protein possessed the NAD-binding glycine-rich Rossmann fold domain ([T–G–
220
X3–G–X–G]) (position 13-20; where X is any amino acid) and the active site residues (Asn112,
221
Ser140, Tyr153 and Lys157) highly conserved among SDRs (Fig. 1). Furthermore, the deduced
222
protein product had a sequence identity of ~25% (~41% similarity) with Zymomonas mobilis
223
RDH [24], the only recombinant RDH reported so far. Members of the SDR protein family are
224
known to share low overall sequence identity of 15 to 30% despite the common fold that is
225
highly conserved [9]. Thus, based on these bioinformatics, the gene from E. aerogenes was
226
annotated as a putative rdh, suggesting that it might encode an RDH. The putative rdh gene from
227
E. aerogenes, with a total length of 729 base pairs (bp), encodes a polypeptide of 242 amino
228
acids, with a calculated molecular mass of 25.8 kDa.
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3.2. Heterologous expression and purification of EaRDH
12 Page 12 of 35
A dehydrogenase assay carried out with extracts of E. coli BL21-CodonPlus (DE3)-RIL
232
harboring pET 28(a)–EaRDH revealed the presence of a high level of RDH activity compared
233
with the control E. coli BL21-CodonPlus (DE3)-RIL cells harboring empty circular plasmid pET
234
28(a). The recombinant EaRDH enzyme was purified to electrophoretic homogeneity from the
235
culture supernatant by Ni-affinity column chromatography.
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3.3. Determination of molecular mass and quaternary structure
238
The subunit molecular mass of the enzyme was 25.8 kDa, as determined by PAGE in the
239
presence of SDS (Fig. 2A). Gel filtration chromatography on a Sephacryl S–300 high-resolution
240
column resulted in the elution of EaRDH as a symmetrical peak between aldolase and
241
conalbumin, corresponding to a molecular weight of approximately 110 kDa (Fig. 2B). These
242
results indicate that the enzyme migrates as a tetramer in gel filtration chromatography and exists
243
as an active tetramer in solution as well, which is typical of other RDHs [6, 25, 26].
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3.4. Optimum pH and temperature
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The effect of pH on enzyme activity is shown in Fig. 3A. Maximal activity was achieved under
247
high alkaline condition at pH 11.0 (pH above 10.0), consistent with the notion that an ionized
248
Tyr153 (pKa = 10) is involved in catalysis. Similar pH dependence has been observed for other
249
members of the sugar alcohol dehydrogenase group [27]. The optimal temperature for the
250
dehydrogenation reaction was found to be 45°C (Fig. 3B). The enzyme retained more than 90%
251
of the maximum activity when assayed at 50°C, with more than 85% retained at 55°C, and more
252
than 80% at 60°C (Fig. 3B). Supplementary Fig. S1 shows the percentage of residual activity vs.
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incubation time, which followed first-order decay with half-lives of 25.7, 15.3, 7.5, 3.1, and 0.45
254
h at 40°C, 45°C, 50°C, 55°C, and 60°C, respectively.
255
3.5. Substrate and coenzyme specificity
257
The characterization of EaRDH as an RDH allowed the investigation of its substrate specificity
258
for various polyols. EaRDH was specific for ribitol and did not show any activity towards other
259
polyols. To the best of our knowledge, this is the first report of a RDH showing ribitol-limited
260
specificity, unlike other RDHs characterized previously exhibiting broad substrate specificities
261
(see Table 1). A measurement with ribitol as the substrate showed that EaRDH was exclusively a
262
NAD+-dependent enzyme showing essentially no activity with NADP+.
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3.6. Effects of metal ions and ICP-MS analysis
265
The EaRDH enzyme was purified followed by extensive dialysis in the presence of 10 mM
266
EDTA. As shown in Table 2, we found that the enzyme activity was mostly lost (~97%) after
267
EDTA treatment. This indicates that EaRDH is a metal-dependent enzyme. The activity of the
268
EaRDH enzyme was completely inhibited by Hg2+ and partially activated by Fe3+. Almost
269
complete recovery of enzyme activity was observed in the presence of 1 mM K+ (96%) and Cu2+
270
(90%). EaRDH activity was significantly stimulated by Ba2+ or Mg2+. However, the highest level
271
of activation was achieved using Ca2+ (5 mM). Ca2+ enhanced the activity about 2.45-fold. The
272
enhancement in activity of EaRDH was found to be dependent on the presence of divalent metal
273
ions in the following order: Ca2+ >> Ba2+ >> Mg2+ >> Mn2+≈ Co2+.
274 275
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To investigate the presence of divalent metal ions in purified EaRDH and assess the ability of EaRDH to bind metal ions, the recombinant protein was subjected to ICP-MS analysis.
14 Page 14 of 35
Based on the apparent molecular mass of 25.8 kDa, 1 μmol of enzyme contained 0.50 ± 0.04
277
μmol of Mg2+. The ICP-MS data provided support for 1 metal binding site per dimer, as
278
observed in other SDRs such as dTDP-6-deoxy- L-lyxo-4-hexulose reductase [28], xylitol
279
dehydrogenase [29], and galactitol dehydrogenase [19]. The concentrations of Mn2+, Zn2+, Ba2+,
280
Ni2+, and Sn2+ were below the detection limit, and no appreciable amount of Ca2+ was detected.
281
Mg2+ enzymes have often been substituted with Ca2+/Cd2+ because of their similar electronic
282
properties [30]. In addition, the coordination chemistry of Ca2+ is closely related to Mg2+. At
283
coordination number (CN) = 6, the ionic radii of Ca2+, Cd2+, and Mg2+ are 1.00, 0.95, and 0.72 A,
284
respectively, whereas at CN = 8 they are 1.12, 1.10, and 0.89 A, respectively [31]. Without a
285
detailed study of metal binding in EaRDH through crystallography or NMR studies, it is not
286
possible to explain why Ca2+ is better than Mg2+ at restoring EaRDH activity.
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3.7. Kinetic parameters of EaRDH
289
Initial velocities were determined in the standard assay mixture at pH 11.0. Ribitol had
290
hyperbolic saturation curves and the corresponding double-reciprocal plots were linear (data not
291
shown). Fig. 4 shows kinetic parameters for EaRDH activity with increasing ribitol
292
concentrations from 0.25 to 300 mM. Maximum enzyme activity was obtained with a ribitol
293
concentration of about 200 mM under the given experimental conditions. The Km values of
294
EaRDH for ribitol and NAD+ were found to be 10.3 and 0.16 mM, respectively (Table 1). The
295
Vmax and kcat/Km values were 185 U mg-1 and 30.9 s-1 mM-1, respectively. The observed activity
296
of EaRDH (185 U mg-1) was the highest of any reported RDH to date (Table 1). The detailed
297
kinetic studies for only two previously characterized RDHs from Klebsiella aerogenes evolved
298
strain A and Z. mobilis have been reported so far. The kcat/Km,Ribitol value (30.9 s-1 mM-1 ) for
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EaRDH was the highest compared with the kcat/Km,Ribitol of 28.7 s-1 mM-1 and 0.41 s-1 mM-1 for
300
KaRDH [32] and ZmRDH [24], respectively. Although a number of sequences for characterized
301
and putative RDHs are present in the NCBI database, detailed quantitative kinetic studies are
302
lacking.
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3.8. Homology modeling of EaRDH
305
The overall secondary structure of the modeled EaRDH enzyme was similar to that of CAD from
306
S. clavuligerus and GatDH from R. sphaeroides D (Supplementary Fig. S2). The generated
307
EaRDH model was validated using the validation tool PROCHECK [33] in order to confirm the
308
consistency of the model. The Profile-3D score of the model was 93.21 against a maximum
309
expected score of 106.72, which compares well to the scores of 109.47 and 112.5 for the
310
coordinates from 2JAH and 2WSB. The built model was also evaluated by superimposing it onto
311
the template crystal structures and the obtained RMSD values were 0.44 Å and 0.6 Å with
312
respect to 2JAH and 2WSB, based on the Cα atoms (Supplementary Fig. S3). The structural
313
model of EaRDH obtained by molecular modeling consists of a classical α/β Rossmann fold
314
pattern commonly found in the SDR family [10, 34]. Upon superimposition, the coenzyme-
315
binding motif and the active site residues of EaRDH superimposed well upon the coenzyme-
316
binding motif and the catalytic residues of S. clavuligerus CAD and R. sphaeroides D GatDH,
317
respectively (Supplementary Fig. S4). Despite low sequence homology among SDR family
318
members, motifs that define classical SDRs are apparent in EaRDH. Apart from the nucleotide-
319
binding motif and the active tetrad of Asn112–Ser140–Tyr153–Lys157, the EaRDH enzyme
320
possesses the conserved but slightly modified Ala87–Asn88–Ala89–Gly90 motif (sequence
321
important for the stabilization of the central β-sheet), a singular Asp60 (residue responsible for
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the stabilization of adenine ring pocket), and a Pro183–Gly184 motif (residues determining
323
reaction direction), followed by a conserved Thr188 (residue responsible for the H-bonding to
324
carboxamide of nicotinamide ring) [10, 11].
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3.9. Cofactor and substrate docking
327
In order to investigate the molecular basis responsible for the unique substrate specificity, we
328
constructed a structural model of EaRDH and docked the cofactor (NAD+) and the substrate
329
(ribitol) into the active site to mimic the interactions between the enzyme, cofactor, and substrate
330
(Supplementary Fig. S5). The axis of the NAD+ ran roughly parallel to the β-sheet and
331
perpendicular to the 6 Rossmann fold helices. The NAD+ was anchored to the enzyme with the
332
nicotinamide ring in the syn conformation, which is stabilized in this conformation by an
333
intramolecular hydrogen bond between the nicotinamide and the pyrophosphate. The coenzyme-
334
binding site is formed by residues 13–20, 38–40, 60–64, 88–90, and 188 (residues numbered
335
with respect to EaRDH). The nicotinamide ribose group is in a cleft surrounded by the conserved
336
catalytic residues Asn112, Tyr153, and Lys157. The loop formed by residues 13–20 is the well-
337
known glycine-rich motif that characterizes many dehydrogenases because its composition and
338
conformational flexibility facilitate interaction with the coenzyme. Near the carboxyl-terminal
339
edge of the dinucleotide fold, bounded on one side by the si-face of nicotinamide, a rather
340
narrow groove serves as the binding site of the substrate (Fig. 5). Because of this narrow width
341
of the groove, the substrate must slip in and bind with its backbone nearly parallel to the
342
nicotinamide plane such that the C2 carbon of the substrate is positioned directly below the C4
343
carbon of the cofactor (Fig. 5A). Upon substrate docking, the C2 hydroxyl of ribitol, which is
344
oxidized by the enzyme to a carbonyl, hydrogen bonds with the catalytic base Tyr153 of
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17 Page 17 of 35
EaRDH. Ribitol, due to its stereochemical arrangement, could slide into this narrow groove,
346
providing all the favorable interactions to ensure the oxidation of ribitol at C2 (Fig. 5B). The C2
347
carbon, which transfers its hydride to NAD+ during the course of the reaction, has a distance of
348
3.6 Å to the nicotinamide C4, suitable for hydride transfer. An attempt to dock other polyol
349
substrates into the narrow substrate-binding pocket of EaRDH resulted in collisions between
350
molecules or a lack of feasible hydrogen bonding between the protein and the cofactor.
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We have modeled a structure of ZmRDH, a broad-specificity RDH, in order to compare
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substrate binding sites of EaRDH and ZmRDH. ZmRDH is the only characterized RDH with
353
known sequence available in NCBI. A total of 28 amino acid residues including the active-site
354
residues were located within the 4.5Å radius of the substrate binding pocket (SBP) of both
355
ZmRDH and EaRDH. Upon alignment, the substrate binding sites of ZmRDH and EaRDH
356
shared only 21.4% of sequence identity (Supplementary Fig. S6), mostly due to the active site
357
residues of Asn112–Ser140–Tyr153–Lys157 (numbered with respect to EaRDH). Without
358
thorough determination of the role of each SBP residue of EaRDH, it is difficult to suggest the
359
molecular determinants of the narrow substrate specificity of EaRDH. The substrate binding site
360
cavities of EaRDH and ZmRDH exhibited volumes of 190Å3 and 256Å3, respectively. The shape
361
and volume of the cavities likely affect the substrate selectivity, and this, in turn, reflects
362
differences in the structure of EaRDH and ZmRDH as evident from the 3D structures (Fig. 6A
363
and B). The binding site cavity of ZmRDH is larger in the vicinity of the catalytic site residues
364
(Asn131–Ser157–Tyr170–Lys174) as compared to the smaller cavity volume of EaRDH. The
365
larger binding site cavity of ZmRDH can accommodate various substrate molecules because of
366
its large size, whereas the smaller EaRDH binding site cavity is not likely to allow the entry of
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18 Page 18 of 35
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various polyol substrates. These findings indicate that the selection of substrate by the narrow
368
substrate binding pocket of EaRDH is more restricted than in previously reported ZmRDH.
369
4. Conclusion
371
In conclusion, we have cloned, expressed, and characterized a highly active RDH from E.
372
aerogenes. The purified recombinant protein showed RDH activity, confirming the identity of
373
the protein. The bio-physiochemical properties and homology modeling studies strongly affirm
374
that EaRDH is a member of the SDR family. The characterized RDH shares common
375
biochemical properties with previously known RDHs, but differs in enzymatic kinetics and
376
substrate specificity. EaRDH showed the highest turnover rate and catalytic efficiency among all
377
previously characterized RDHs. A detailed interpretation of the marked preference of EaRDH
378
for ribitol as its sole substrate and its high activity will rely on 3D structural analysis, which is
379
our future direction of study.
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Acknowledgements
382
This work was supported by the Energy Efficiency & Resources Core Technology Program of
383
the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial
384
resource from the Ministry of Trade, Industry & Energy, Republic of Korea (201320200000420).
385
This research was also supported by Basic Science Research Program through the National
386
Research Foundation of Korea funded by the Ministry of Education, Science and Technology
387
(NRF-2013R1A1A2007561). This work was supported by 2014 KU Brain Pool fellowship of
388
Konkuk University.
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19 Page 19 of 35
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References
390
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bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation
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[7] Wymer N, & Taylor, P., inventors; Zuchem, Inc. assignee. Production of L-ribose and other rare sugars. United States patent US8642297. 2014 Feb 4. [8] Persson B, Kallberg Y. Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs). Chemico-Biological Interactions. 2013;202:111-5.
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[9] Kallberg Y, Oppermann U, Persson B. Classification of the short-chain
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dehydrogenase/reductase superfamily using hidden Markov models. FEBS J.
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dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact. 2003;143-144:247-
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[12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of
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protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. [13] Singh RK, Tiwari MK, Kim D, Kang YC, Ramachandran P, Lee JK. Molecular cloning and
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enzymatic pretreatment of biomass. Appl Microbiol Biotechnol. 2012.
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[14] Lee KM, Kalyani D, Tiwari MK, Kim TS, Dhiman SS, Lee JK, et al. Enhanced enzymatic hydrolysis of rice straw by removal of phenolic compounds using a novel laccase from yeast
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Yarrowia lipolytica. Bioresour Technol. 2012;123:636-45.
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[15] Jagtap SS, Dhiman SS, Kim TS, Kim IW, Lee JK. Characterization of a novel endo-beta-
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1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis. Appl
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[16] Tiwari MK, Singh RK, Singh R, Jeya M, Zhao H, Lee JK. Role of conserved glycine in
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zinc-dependent medium chain dehydrogenase/reductase superfamily. J Biol Chem.
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2012;287:19429-39.
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[17] Sali A, Potterton L, Yuan F, van Vlijmen H, Karplus M. Evaluation of comparative protein modeling by MODELLER. Proteins. 1995;23:318-26. [18] MacKenzie AK, Kershaw NJ, Hernandez H, Robinson CV, Schofield CJ, Andersson I. Clavulanic acid dehydrogenase: structural and biochemical analysis of the final step in the
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biosynthesis of the beta-lactamase inhibitor clavulanic acid. Biochemistry. 2007;46:1523-33.
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[19] Carius Y, Christian H, Faust A, Zander U, Klink BU, Kornberger P, et al. Structural insight
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into substrate differentiation of the sugar-metabolizing enzyme galactitol dehydrogenase
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from Rhodobacter sphaeroides D. J Biol Chem. 2010;285:20006-14.
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[20] Tiwari M, Lee JK. Molecular modeling studies of L-arabinitol 4-dehydrogenase of
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Hypocrea jecorina: its binding interactions with substrate and cofactor. J Mol Graph Model.
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2010;28:707-13.
[21] Moon HJ, Tiwari MK, Singh R, Kang YC, Lee JK. Molecular determinants of the cofactor
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specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase. Appl Environ
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Microbiol. 2012;78:3079-86.
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[22] Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M.
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CHARMM: A program for macromolecular energy, minimization, and dynamics
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[23] Wu G, Robertson DH, Brooks CL, 3rd, Vieth M. Detailed analysis of grid-based molecular
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docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm. J Comput
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Chem. 2003;24:1549-62.
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[24] Moon HJ, Tiwari M, Jeya M, Lee JK. Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis. Appl Microbiol Biotechnol. 2010;87:205-14.
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[25] Muniruzzaman S, Kunihisa Y, Ichiraku K, Izumori K. Purification and characterization of a
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ribitol dehydrogenase from Enterobacter agglomerans strain 221e. Journal of Fermentation
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and Bioengineering. 1995;79:496-8.
460 461
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Purification and subunit structure. Biochem J. 1974;141:693-700.
[27] Lee D, Redfern O, Orengo C. Predicting protein function from sequence and structure. Nat
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[26] Taylor SS, Rigby PW, Hartley BS. Ribitol dehydrogenase from Klebsiella aerogenes.
Rev Mol Cell Biol. 2007;8:995-1005.
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[28] Blankenfeldt W, Kerr ID, Giraud MF, McMiken HJ, Leonard G, Whitfield C, et al. Variation on a theme of SDR. dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD) shows a
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new Mg2+-dependent dimerization mode. Structure. 2002;10:773-86.
467 468
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466
dehydrogenase cosubstrate specificity. Structure. 2006;14:567-75. [30] Dudev T, Lim C. Principles governing Mg, Ca, and Zn binding and selectivity in proteins.
te
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[29] Ehrensberger AH, Elling RA, Wilson DK. Structure-guided engineering of xylitol
Chem Rev. 2003;103:773-88.
[31] Shannon RD. Revised effective ionic radii and systematic studies of interatomic distances in
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464
an
462
469
halides and chalcogenides. Acta Crystallographica Section A: Crystal Physics, Diffraction,
470
Theoretical and General Crystallography. 1976;32:751-67.
471 472 473
[32] Burleigh BD, Rigby PW, Hartley BS. A comparison of wild-type and mutant ribitol dehydrogenases from Klebsiella aerogenes. Biochem J. 1974;143:341-52. [33] Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check
474
the stereochemical quality of protein structures. Journal of Applied Crystallography.
475
1993;26:283-91.
23 Page 23 of 35
476
[34] Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, et al. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem.
478
2002;277:25677-84.
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24 Page 24 of 35
ip t
Optimum temperature
Metal ion
Specific activity (Units/mg)
Klebsiella aerogenes A
11.0
NR
Zn2+
NR
Rhodobacter sphaeroides Si4
10.0
45°C
NR
9.0–10.0
9.5–10.5
NR
Sn2+/ Na2+
NR
ce pt
Gluconobacter oxydans IFO 12528
50–60°C
ed
Enterobacter agglomerans
Km (mM)
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Optimum pH
59
144
Reference
Substrate (Ribitol)
0.66
10.4
Ribitola, xylitol, L-arabitol
[24]
0.08
6.3
Ribitol (100)b, xylitol (21), erythritol (12), D-sorbitol (10), D-arabitol (10)
[33]
32.2
Ribitol (100)c, allitol (66), L-arabitol (56.5), L-mannitol (44.5), xylitol (42.5), L-sorbitol (41), L-talitol (26.5), L-iditol (7), D-sorbitol (5), galactitol (3), erythritol (1)
[23]
1.2
Ribitol (100)d, xylitol (85.6), L-arabitol (79.8), D-arabitol (16.1), erythritol (6.8), D-sorbitol (3.4)
[6]
[22]
This study
2.36
29.2
Substrate specificity (relative % activity)
Coenzyme (NAD+)
M an
Organism
cr
Table 1. Biochemical and kinetic properties of ribitol dehydrogenases from various organisms
0.08
9.5
65°C
Mn2+
5.65
0.18
11.8
Enterobacter aerogenes KCTC 2190
11.0
45°C
Mg2+
185
0.16
10.3
Ribitol (100)f
Ac
Zymomonas mobilis
Ribitol (100)e, D-threitol (105), Lthreitol (98.7), L-arabitol (102), xylitol (91.2), glycerol (89.1), mannitol (84.9), galactitol (55.6), sorbitol (41.1)
NR, Not reported a Relative activity not specified b Values in parentheses indicate relative activity at 150 mM substrate concentration c Values in parentheses indicate relative activity at 10 mM substrate concentration d Values in parentheses indicate relative activity at 100 µM substrate concentration e Values in parentheses indicate relative activity at 100 mM substrate concentration f Values in parentheses indicate relative activity at 200 mM substrate concentration
25 Page 25 of 35
Table 2. Effects of different metal ions on the activity of EaRDH. The EDTA-treated enzyme was assayed in standard assay conditions with 1 mM or 5 mM metal ions. The activity of EaRDH before EDTA treatment was set as 100%. The metal-depleted apoenzyme retained
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only 3% activity relative to the native EaRDH enzyme. Each value represents the mean of
Mg2+
148 ± 4
Mn2+
161 ± 6
Zn2+
139 ± 3
Ca2+
193 ± 7
Fe3+
45.5 ± 4.8
27.2 ± 2.4
2+
90.1 ± 3.1
50.5 ± 6.1
119 ± 2
108 ± 4
0
0
142 ± 3
209 ± 6
95.7 ± 7.0
73.6 ± 3.5
Hg2+ Ba2+
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K+
d
Co2+
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Cu
Relative activity (%) 5 mM 100 ± 4
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Before EDTA treatment
Relative activity (%) 1 mM 100 ± 2
Metal ions
479
cr
triplicate measurements and varied from the mean by not more than 15%.
162 ± 6 109 ± 8
61.9 ± 5.0 245 ± 8
26 Page 26 of 35
479
Figure legends
480
Fig. 1. Multiple sequence alignment of E. aerogenes RDH with selected short-chain
482
reductase/dehydrogenases (SDRs). EaRDH, Enterobacter aerogenes RDH (Accession number
483
YP_004594905.1); GoXDH, Gluconobacter oxydans xylitol dehydrogenase (Accession number
484
JC7939); RsGatDH, Rhodobacter sphaeroides galactitol dehydrogenase (Accession number
485
2WDZ); AbMDH, Agaricus bisporus mannitol dehydrogenase (Accession number 1H5Q);
486
CtHSD, Comamonas testosteroni 3β/17β-hydroxysteroid dehydrogenase (Accession number
487
1HXH); MmCR, Mus musculus carbonyl reductase (Accession number NP_031647.1);
488
BnACPR, Brassica napus enoyl-acyl carrier protein (acp) reductase (Accession number 1EDO);
489
BmGDH, Bacillus megaterium glucose dehydrogenase (Accession number P40288.1); KpBDH,
490
Klebsiella pneumonia meso-2,3-butanediol dehydrogenase (Accession number 1GEG); and
491
HsXR, Homo sapiens xylulose reductase (Accession number NP_057370.1). The four members
492
of the catalytic tetrad are indicated with red boxes. The glycine-rich consensus sequences that
493
have a structural role in coenzyme binding in all SDRs are indicated by the yellow box. Amino
494
acids conserved, semi-conserved, and weakly conserved are displayed in dark blue, blue, and
495
light blue, respectively.
cr
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M
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496
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481
497
Fig. 2. Determination of the molecular mass of E. aerogenes RDH by SDS-PAGE and gel
498
filtration chromatography. (A) Overexpression and purification of recombinant EaRDH. Lane M,
499
molecular mass Marker; Lane 1, soluble fraction of E. coli (control) harboring empty pET 28a;
500
Lane 2, soluble fraction of E. coli harboring recombinant pET 28a–EaRDH; Lane 3, purified
501
recombinant EaRDH. (B) Determination of native molecular mass of EaRDH by gel filtration
27 Page 27 of 35
502
chromatography on a sephacryl S–300 high-resolution column (Amersham). The column was
503
calibrated with standard molecular mass proteins aldolase (158 kDa), conalbumin, (75 kDa),
504
bovine serum albumin (66 kDa), and ovalbumin (44 kDa).
ip t
505
Fig. 3. Effects of pH (A) and temperature (B) on the activity of EaRDH. Enzyme assays were
507
carried out under standard conditions in the presence of 200 mM ribitol. Activities at the optimal
508
temperature and pH were defined as 100%. Each value represents the mean of triplicate
509
measurements and varied from the mean by not more than 10%. 100 mM sodium acetate buffer
510
(filled circles), 100 mM Tris-HCl buffer (open circle), and 100 mM Tris-glycine NaOH buffer
511
(inverted triangle).
an
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506
M
512
Fig. 4. Effect of substrate concentration on the activity of EaRDH. RDH activity of the enzyme
514
was measured in the presence of the indicated concentration of substrate at pH 11.0. The data
515
represent an average of all statistically relevant data with a standard deviation of less than 15%.
te
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516
d
513
517
Fig. 5. Molecular surface representation of the EaRDH model. (A) Stereo view and (B) front
518
view of the molecular surface representation of the substrate binding pocket of the EaRDH
519
model. The active site residues (Asn112, Ser140, Tyr153, and Lys157) of EaRDH are shown in
520
green carbon. Hydrogen bonds and π -π interactions are represented by dashed green and orange
521
lines, respectively. The path of hydride transfer between ribitol C2 and NAD+ C4 is marked with
522
green line. The hydrophobic region, hydrogen bond donor, and hydrogen bond acceptor atoms
523
are depicted in yellow, green, and brown, respectively. For clarity, only the nicotinamide ring of
524
NAD+ is presented.
28 Page 28 of 35
525
Fig. 6. Active site cavities of EaRDH and ZmRDH. The active site cavities were calculated using
527
DS 3.1 and rendered as mesh surfaces. Full views of the (A) EaRDH and (B) ZmRDH models.
528
Active site residues are colored with blue (ZmRDH) and green (EaRDH) colored carbon, and
529
other residues near the active site are colored with white (ZmRDH) and grey colored carbon
530
(EaRDH). NAD+ is shown as sticks with orange carbon and ribitol as ball and sticks with yellow
531
carbon.
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29 Page 29 of 35
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M
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A
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B
Fig. 2 31 Page 31 of 35
M
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A
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B
Fig. 3 32 Page 32 of 35
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Fig. 4
33 Page 33 of 35
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B
d
M
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A
Fig. 5 34 Page 34 of 35
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A
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B
Fig. 6 35 Page 35 of 35