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Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis
Protein Expression and Purification 166 (2020) 105523
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis
T
Muhammad Arifa, Qamar Bashira, Masood Ahmad Siddiquib, Naeem Rashida,∗ a b
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan Department of Chemistry, Balochistan University, Saryab Road, Quetta, 87300, Pakistan
ARTICLE INFO
ABSTRACT
Keywords: Geobacillus thermopakistaniensis Thermophilic Phosphoribosyl anthranilate isomerase TrpF Structural stability
Phosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(ocarboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombinant TrpFGt was highly efficient and stable. The apparent Vmax and Km values were 480 μmol min−1 mg−1 and 1.15 μM, respectively. The half-life of the enzyme was 90 min at 60 °C. Apart from thermostability, TrpFGt was highly stable against protein denaturants such as urea. There was no significant change in activity even after treatment with 8 M urea. To the best of our knowledge, TrpFGt, is the most active and stable phosphoribosyl anthranilate isomerase characterized to date and this is the first characterization of TrpF from the genus Geobacillus.
1. Introduction Five, structurally and functionally distinct enzymes are involved in the biosynthesis of L-tryptophan from chorismic acid. These enzymes are encoded by seven different genes. Two genes, trpE and trpG, encode anthranilate synthase (EC: 4.1.3.27, AS) which catalyzes the reversible conversion of chorismate and L-glutamine to anthranilate, pyruvate and L-glutamate, the first step in the tryptophan biosynthesis pathway. In the second step, anthranilate phosphoribosyl transferase (EC: 2.4.2.18, PRT), which is encoded by the trpD gene, catalyzes the reversible conversion of anthranilate to 5-phosphoribosyl-anthranilate. 5Phosphoribosyl-anthranilate, in the next step, is converted to carboxyphenylamino-deoxyribulose 5-phosphate by phosphoribosyl anthranilate isomerase (EC: 5.3.1.24, PRAI), which is encoded by the trpF gene. The product of this reaction is converted to indole-3-glycerolphosphate by indole-3-glycerol-phosphate synthase (EC: 4.1.1.48, InGPS) encoded by the trpC gene. The fifth enzyme involved in the biosynthesis of tryptophan is tryptophan synthase (EC: 4.2.1.20, TS) encoded by the trpB and trpA genes. This enzyme catalyzes the final two steps in the biosynthesis of tryptophan. The product of trpA catalyzes the reversible cleavage of indole-3-glycerol-phosphate to form indole and glyceraldehyde 3-phosphate, whereas the product of trpB catalyzes the irreversible condensation of indole and serine to form tryptophan [1]. ∗
The structural organization of the third enzyme, phosphoribosyl anthranilate isomerase, of the tryptophan synthesis pathway differs widely among microorganisms. It is a monofunctional enzyme of a single polypeptide chain in Acinetobacter calcoaceticus [2], Bacillus subtilis [3], Pseudomonas putida [4], and Saccharomyces cerevisiae [5]. However, in Aerobacter aerogenes [6], Escherichia coli [7], Salmonella typhimurium [8], and Serratia marcescens [9], it is found together with indole-3-glycerol-phosphate synthase in the same polypeptide chain. This enzyme has been characterized from all the three domains of life including eukaryotes [10–12], archaea [13–15], and bacteria [16–18]. Phosphoribosyl anthranilate isomerase has been characterized from several bacteria however, to the best of our knowledge, there is no report available from the genus Geobacillus. We are interested in the tryptophan metabolism in thermophilic microorganisms. We have previously characterized anthranilate phosphoribosyltransferase and phosphoribosyl anthranilate isomerase from the hyperthermophilic archaeon Thermococcus kodakarensis [15,19], and indole-3-glycerol-phosphate synthase from the hyperthermophilic archaeon Pyrococcus furiosus [20]. Recent ease in genome sequencing has piled up the amino acid sequences of uncharacterized enzymes including those involved in tryptophan synthesis. The genome sequence of Geobacillus thermopakistaniensis, a thermophilic bacterium, has been determined [21]. The genome sequence analysis revealed the presence of a phosphoribosyl anthranilate isomerase homologue; however, the
Corresponding author. E-mail addresses: [email protected], [email protected] (N. Rashid).
https://doi.org/10.1016/j.pep.2019.105523 Received 26 August 2019; Received in revised form 18 October 2019; Accepted 23 October 2019 Available online 29 October 2019 1046-5928/ © 2019 Elsevier Inc. All rights reserved.
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enzyme has not been functionally characterized yet. In this manuscript we describe molecular cloning and characterization of this enzyme as a first step of understanding of the tryptophan metabolism in G. thermopakistaniensis.
against their retention volumes. The standard proteins used were lysozyme (14 kDa), proteinase K (29 kDa), ovalbumin (44 kDa), bovine serum albumin (66 kDa), and alcohol dehydrogenase (147 kDa). 2.5. Activity assay
2. Materials and methods
Enzyme activity of TrpFGt was determined by a coupled enzyme assay system by using the thermostable TrpD from T. kodakarensis (prepared in our lab) as shown in the scheme below.
2.1. Reagents, vectors and bacterial strains The reagents used in this study were of analytical grade and purchased either from Thermo Fisher Scientific, USA or Sigma-Aldrich, USA or Fluka Chemicals, UK. Restriction enzymes and DNA purification kits were purchased from Thermo Fisher Scientific. The gene specific primers were chemically synthesized from Macrogen, Korea and expression vector pET-21a was purchased by Novagen®, USA. The genetically engineered E. coli strains DH5α and BL21- CodonPlus (DE3)RIL were procured from Stratagene, USA.
Anthranilate + Phosphoribosyl pyrophosphate (PRPP ) anthranilate (PRA) + pyrophosphate (PPi) Phosphoribosyl anthranilate (PRA) 1
TrpFGt
1
(o
deoxyribulose 5
Phosphoribosyl
carboxphenyla min o) phosphate (CDRP )
As the substrate for TrpFGt is highly unstable at higher temperatures therefore a continuous coupled enzyme activity assay was performed. For the preparation of the substrate, the initial assay mixture, 1.95 mL, containing 0.1 M Tris-HCl (pH 8.0), 33 μM anthranilate, 100 μM ZnCl2 and 10 μg of TkTrpD (an anthranilate phosphoribosyl transferase from T. kodakarensis) was incubated at 60 °C (optimal temperature for TkTrpD) for 5 min. The reaction was initiated with the addition of phosphoribosyl pyrophosphate (1 mM) and 10 μg of TrpFGt. After 3 min of incubation at 60 °C, the reaction was stopped by quenching in ice water. The decrease in the amount of anthranilate was measured with a fluorescent spectrophotometer. The activation and emission wavelengths were set at 310 nm and 390 nm, respectively. By using a standard curve, the amount of carboxyphenylamino-deoxyribulose 5-phosphate produced per min was calculated (production of 1 μmol of carboxyphenylamino-deoxribulose 5-phosphate is equivalent to consumption of 1 μmol of anthranilate).
2.2. Cloning and expression constructs The gene encoding phosphoribosyl anthranilate isomerase from G. thermopakistaniensis, trpFGt (accession # ESU71245) was amplified by polymerase chain reaction (PCR) by utilizing genomic DNA of G. thermopakistaniensis as template and commercially synthesized forward ( 5′-CATATGATTCGAATTAAATATTGCGGCAACCG) and reverse (5′-TCA TCGACACCCGTGATGTT) primers. An NdeI site (underlined sequence) was introduced in the forward primer. The PCR amplified DNA fragment was ligated in pTZ57 R/T cloning vector. The resulting recombinant vector was designated as pTZ-TrpFGt. The TrpFGt gene was excised from pTZ-TrpFGt using NdeI (incorporated in the forward primer) and BamHI (present in multiple cloning site of pTZ57 R/T). The excised gene was sub-cloned in pET-21a expression vector utilizing the same restriction sites. The resulting recombinant plasmid was denoted as pET-TrpFGt.
2.6. Effect of chemical denaturants The effect of chemical denaturants on the stability of TrpFGt was examined by incubating the protein samples with various final concentrations of urea (0–8 M) and guanidinium hydrochloride (0–6 M) for 45 min at room temperature. The residual activities of the samples were examined as described above. In addition to the residual activities, these samples were analyzed by circular dichroism (CD) spectroscopy using a Chirascan™-plus CD Spectrometer (Applied Photophysics). CD spectra were recorded in the far-UV region (200–280 nm).
2.3. Production and purification of TrpFGt The recombinant plasmid, pET-TrpFGt, was used to transform E. coli BL21-CodonPlus (DE3)-RIL cells and cultivated at 37 °C in Luria–Bertani broth containing 100 μg/mL ampicillin till the optical density, at 660 nm, reached to 0.4–0.6. The gene expression was induced by the addition of 0.2 mM isopropyl-β-d-1- thiogalactopyranoside for 5–6 h. The cell pellet (~3 g wet weight from 1 L culture), obtained by centrifugation, was resuspended in 50 mM Tris-HCl buffer (pH 8) and lysed by sonication. Soluble and insoluble fractions were separated by centrifugation at 20,000 × g. The soluble fraction was subjected to heat-treatment at 60 °C for 25 min and centrifuged at 20,000 × g to remove heat-denatured proteins of E. coli. ÄKTA Purifier system (GE Healthcare) was employed for further purification of recombinant TrpFGt using a HiTrap® Q anion exchange column (GE Healthcare). Elution of the proteins bound to the column was performed by using a linear gradient of 0–1 M NaCl. The fractions containing a significant amount of TrpFGt were pooled, dialyzed against 50 mM Tris-HCl (pH 8.0) and applied to Resource™ Q, another anion exchange column (GE Healthcare). The bound proteins were eluted in a similar way using a linear gradient of 0–1 M NaCl. Quantification of purified TrpFGt was done spectrophotometrically using Bradford reagent (BioRad) and purity of the protein was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2.7. Structural modeling The three-dimensional structural model of TrpFGt was obtained from I-TASSER [22]. The model obtained was evaluated by its C-score. The structure obtained was compared with the previously reported crystal structures of phosphoribosyl anthranilate isomerase from Thermotoga maritima (1NSJ) and E. coli (1PII) using PyMOL software (https:// pymol.org/). 3. Results The gene, trpFGt, encoding phosphoribosyl anthranilate isomerase in G. thermopakistaniensis comprises 642 nucleotides corresponding to a polypeptide of 213 amino acids. The calculated molecular weight and isoelectric point of the protein were 23,090 Da and 6.06, respectively. A homology search demonstrated that TrpFGt was 100% identical in primary structure with an uncharacterized phosphoribosylanthranilate isomerase from Geobacillus zalihae (accession # WP_081132668). Among the characterized enzymes, highest identity of 48.5% was found with the counterpart from Bacillus subtilis (WP_004429772), followed by 35% from Pseudomonas putida (WP_065861173) and 32.5% from E. coli (AIT70912). A comparison of sequences of the characterized
2.4. Gel filtration chromatography Gel filtration chromatography was performed by using a Superdex 200 Increased 10/300 column (GE Healthcare) equilibrated with 100 mM NaCl in 10 mM Tris–HCl pH 7.5. The standard curve was obtained by plotting the log of molecular weights of standard proteins 2
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Fig. 1. Alignment of six conserved regions found in the characterized TrpF enzymes from bacteria and archaea. The amino acid residues involved in substrate binding and catalysis in TrpF from T. kodakarensis and T. thermophilus are shown in bold and bold-underlined, respectively. Names at the left-hand side indicate the source organism from which the sequence originated. The numbers show the position of the amino acid in the protein sequence. The accession numbers of the sequences are: B. subtilis (BSU22650), Chlamydia trachomatis (B0BBV9), E. coli (AGW08587), Haloferax volcanii (P52563), T. thermophilus (YP_143342), T. maritima (AAD35232), Acinetobacter calcoaceticus (AQZ83080), Pseudomonas putida (Q88LE0), T. kodakarensis (Q9YGB1) and P. furiosus (Q8U092).
phosphoribosyl anthranilate isomerases from bacteria and archaea (Fig. 1) demonstrated that thermostable enzymes contain a higher number of Glu and Lys residues, and a lower percentage of Gln and His in agreement with the literature [20,23]. From the sequence alignment, six highly conserved regions found in bacterial and archaeal enzymes were figured out (Fig. 1). The amino acid residues involved in catalysis and substrate binding in the enzyme from T. kodakarensis [15], completely conserved in the characterized enzymes from bacteria and archaea, are shown Fig. 1.
3.3. Effect of metal ions Addition of 1 mM EDTA in the assay mixture drastically decreased the activity which indicated that TrpFGt activity might be dependent on metal ions. Therefore, we examined the effect of various metal ions at a final concentration of 100 μM. A two-fold enhancement in the activity was found in the presence Zn+2 (Fig. 4). Presence of Ca+2 and Mg+2 also enhanced the activity but not significantly. Enhancement of activity in the presence of Zn+2 is a unique feature of TrpF. All the previously characterized phosphoribosyl anthranilate isomerases are reported to be activated in the presence of Mg+2.
3.1. Biosynthesis and purification of TrpFGt
3.4. Structural stability against temperature and denaturants
Induction of host E. coli cells carrying pET-TrpGt plasmid resulted in production of recombinant protein in soluble and active form at high levels, almost 35% of E. coli proteins. Heat-treatment, the initial purification step, removed most of the heat-labile proteins of the host. Ionexchange chromatography of the soluble part, after heat treatment, resulted in an apparent homogeneous protein band on SDS-PAGE (Fig. 2A). Various steps involved in purification of recombinant TrpFGt and respective enzyme activities at each step are summarized in Table 1. When analyzed by gel filtration, TrpFGt eluted at a retention volume of 16.9 mL, equivalent to a molecular weight of ~50 kDa (Fig. 2B). This result indicated that recombinant TrpFGt existed in a dimeric form in solution, similar to the thermostable counterparts from T. maritima [25] and T. thermophilus [18].
As the optimal temperature for TrpFGT activity was 60 °C, we therefore measured the stability of the enzyme at this temperature. TrpFGT was quite stable at this temperature with a half-life of 90 min (Fig. 5A). However, at higher than 60 °C, the activity reduced drastically. We further examined the effect of denaturants by incubating TrpFGt, at room temperature for 45 min, in the presence of 0–8 M urea or 0–6 M Gdn-HCl (final concentration) and measuring the residual activity. There was no detectable effect even at 8 M urea. However, the activity gradually decreased with the increasing concentration of GdnHCl. There was more than 40% decrease in activity even at 2 M. The activity was completely inhibited at 6 M (Fig. 5B). Apart from activity measurements, these protein samples were analyzed by CD spectrometry. There was no significant change in CD spectra of the samples incubated in the presence or absence of various concentrations of urea (Fig. 5C). However, there were significant changes in CD spectra of TrpFGt samples incubated in the presence of Gdn-HCl (Fig. 5D), which matched the residual activities of these samples.
3.2. Optimal temperature and pH When the reaction catalyzed by TrpFGt was carried out at various temperatures (20–80 °C), the enzyme activity increased gradually with the increase in temperature till the highest at 60 °C (Fig. 3A). Similarly, when we measured the activity by varying the pH from 5.5 to 9.5 and keeping the temperature unchanged at 60 °C, the highest activity was found at pH 8.0 (Fig. 3B).
3.5. Kinetic parameters Measurements of kinetic parameters were performed by plotting the initial reaction velocities against various substrate concentrations. The reaction catalyzed by TrpFGt followed Michaelis-Menten kinetics 3
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Fig. 3. Effect of temperature and pH on enzyme activity of TrpFGt. A) Effect of temperature. Activity assays were performed at various temperatures ranging from 30 to 80 °C at pH 8.0 in 50 mM Tris-HCl. B) Effect of pH. Activity assays were performed at 60 °C and various pH. Two buffer systems, Na-phosphate buffer pH 6.0–7.5 (squares) and Tris-HCl buffer pH 7.5–9.5 (circles), were used. Error bars indicate standard deviation.
Fig. 2. Purity and molecular weight determination. A) Coomassie brilliant blue stained 15% SDS-PAGE showing the purified recombinant TrpFGt. Lane M, molecular weight marker; lane 1, purified TrpFGt (10 μg). B) Plot of log of molecular weight of the standards used for gel filtration chromatography against Kav, calculated from their retention volumes, for molecular weight and subunit determination. Filled circles show the position of molecular weight standards while the open circle shows the position of TrpFGt. Error bars indicate standard deviation. Table 1 Purification of recombinant TrpFGt. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification-fold
Yield (%)
Total cell lysate Heat treatment HiTrap QFF Resource Q
60 17 11 8
5023 4196 3677 3176
84 247 334 397
1 3 4 4.7
100 84 73 63
Fig. 4. Effect of various metal cations on TrpFGt activity. Reactions were performed at 60 °C and pH 8.0. Chloride salt of each metal cation was used at a final concentration of 100 μM. EDTA, when used, was 1 mM. Each measurement is the average value of three independent experiments.
4
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Fig. 5. Structural stability of TrpFGt against temperature and denaturants. A) Thermostability of TrpFGt. The protein was incubated at 60 °C for various time intervals and residual activity was measured at 60 °C and pH 8.0 in Tris-HCl buffer. B) Effect of denaturants. TrpFGt was incubated at room temperature for 45 min in the presence of various concentrations of urea (circles) and guanidine-HCl (squares) and residual activity was measured. C) Structural stability against urea. Before measuring the CD spectra, TrpFGt was incubated at room temperature for 45 min in the presence of 0 (open squares), 2 (filled squares), 4 (filled triangles), and 8 M (open circles) urea (final concentration). D) Structural stability against guanidinium-HCl. Before measuring the CD spectra, TrpFGt was incubated at room temperature for 45 min in the presence of 0 (squares), 4 (circles) and 6 M (triangles) guanidinium-HCl. CD spectra were recorded in the far-UV region (200–280 nm).
(Fig. 6A). The apparent Vmax and Km values were 480 ± 10 μmol min−1 mg−1 and 1.15 ± 0.05 μM, respectively. The Vmax exhibited by TrpFGt is the highest among the characterized phosphoribosyl anthranilate isomerases. TrpFGt exhibited a kcat value of 185 s−1 subunit−1. The low Km and the high kcat values of TrpGGt, compared to its bacterial counterparts, indicate that catalytically it is a highly efficient enzyme. The reaction catalyzed by TrpFGt at various temperatures followed a linear trend till 60 °C. By using the rates of reaction at various temperatures, an Arrhenius plot (lnk vs. 1/T) was used to calculate the activation energy which came out to be 9.02 ± 0.5 kJ mol−1 (Fig. 6B). Activation enthalpy and entropy calculated from Eyring–Polanyi plot (lnk/T vs. 1/T) were and –116 ± 1 J mol−1 K−1, respectively 16.5 ± 0.5 kJ mol−1 (Fig. 6C).
substrate binding site. 4. Discussion Recent advancements have in DNA sequencing has piled up the amino acid sequences of the uncharacterized proteins. A protein data base search provided by National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/protein/?term=phosphoribosyl anthranilate + isomerase) revealed more than ninety thousand protein sequences annotated as phosphoribosylanthranilate isomerases. However, a very few of them have been experimentally verified to be true phosphoribosylanthranilate isomerase. Experimental examination of these uncharacterized proteins is expected to lead the discovery of novel enzymes. We have previously characterized a few enzymes involved in tryptophan metabolism in T. kodakarensis [15,19] and P. furiosus [20]. We have described here, TrpFGt, a phosphoribosylanthranilate isomerase from G. thermopakistaniensis, which was produced in E. coli in soluble and highly active form. A novel feature of TrpFGt was its highest activity, 480 μmol min−1 mg−1, among the characterized counterparts. Similarly, the kcat/Km exhibited by TrpFGt is the highest among the characterized phosphoribosylanthranilate isomerases, indicating that it is a highly efficient enzyme. TrpFGt exhibited highest activity in the presence of Zn+2 whereas the enzyme from E. coli exhibited highest activity in the presence of Mg+2. TrpFGt is the only
3.6. Structural analysis The structural model obtained from I-TASSER had C-score of 1.25, signifying high confidence of the predicted model. TrpFGt structural model was superimposed with the previously reported crystal structures of its counterparts from T. maritima and E. coli (Fig. 7). The TrpFGt structure shows more similarity with the one from T. maritima (RMSD of 0.75) as compared to that of E. coli (RMSD of 1.20). However, it has notable differences from the other two structures, particularly near the 5
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Fig. 7. Superimposition of the three-dimensional structure of TrpFGt (red) on the crystal structures of phosphoribosyl anthranilate isomerases from T. marimtima (blue) and E. coli (green). The residues involved in substrate binding and catalysis are labeled and shown as sticks. The figure was drawn in PyMOL (https://pymol.org/).
proteins from hyperthermophilic archaea and thermophilic bacteria are reported to maintain their native structures, and hence the enzyme activities, in the presence of these denaturants [26-28]. We also examine the activity of TrpFGt after incubating with various concentrations of urea or guanidinium chloride and found that there was no significant loss in activity even after incubating in 8 M urea. However, the activity was reduced to 20% in the presence of 6 M guanidinium chloride. This could be due to the fact that guanidinium chloride is a salt as well as a denaturant whereas urea is an uncharged molecule, hence, has less ionic strength effects. The predicted model of TrpFGt indicates that the binding site has structural differences with its counterparts from T. maritima and E. coli that may account for its unique characteristics. In conclusion, the results obtained in this study demonstrate that TrpFGt is highly resistant to urea, a protein denaturant. It exhibits the highest ever reported phosphoribosyl anthranilate isomerase activity. Another unique feature of the enzyme is its activation by Zn+2. Further studies are underway to elucidate the structure function relationship of the enzyme. Declaration of competing interest The authors declare no conflict of interest.
Fig. 6. Determination of kinetic parameters. A) Substrate-dependent enzyme activity of TrpFGt. The data was used to fit the Michaelis–Menten equation to obtain the apparent Km and Vmax values. B) Arrhenius plot (lnk versus reciprocal absolute temperature) for the activation energy of TrpFGt. C) Eyring–Polanyi plot (ln(k/T) versus reciprocal absolute temperature) for calculation of enthalpy and entropy of the reaction catalyzed by TrpFGt.
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis
T
Muhammad Arifa, Qamar Bashira, Masood Ahmad Siddiquib, Naeem Rashida,∗ a b
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan Department of Chemistry, Balochistan University, Saryab Road, Quetta, 87300, Pakistan
ARTICLE INFO
ABSTRACT
Keywords: Geobacillus thermopakistaniensis Thermophilic Phosphoribosyl anthranilate isomerase TrpF Structural stability
Phosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(ocarboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombinant TrpFGt was highly efficient and stable. The apparent Vmax and Km values were 480 μmol min−1 mg−1 and 1.15 μM, respectively. The half-life of the enzyme was 90 min at 60 °C. Apart from thermostability, TrpFGt was highly stable against protein denaturants such as urea. There was no significant change in activity even after treatment with 8 M urea. To the best of our knowledge, TrpFGt, is the most active and stable phosphoribosyl anthranilate isomerase characterized to date and this is the first characterization of TrpF from the genus Geobacillus.
1. Introduction Five, structurally and functionally distinct enzymes are involved in the biosynthesis of L-tryptophan from chorismic acid. These enzymes are encoded by seven different genes. Two genes, trpE and trpG, encode anthranilate synthase (EC: 4.1.3.27, AS) which catalyzes the reversible conversion of chorismate and L-glutamine to anthranilate, pyruvate and L-glutamate, the first step in the tryptophan biosynthesis pathway. In the second step, anthranilate phosphoribosyl transferase (EC: 2.4.2.18, PRT), which is encoded by the trpD gene, catalyzes the reversible conversion of anthranilate to 5-phosphoribosyl-anthranilate. 5Phosphoribosyl-anthranilate, in the next step, is converted to carboxyphenylamino-deoxyribulose 5-phosphate by phosphoribosyl anthranilate isomerase (EC: 5.3.1.24, PRAI), which is encoded by the trpF gene. The product of this reaction is converted to indole-3-glycerolphosphate by indole-3-glycerol-phosphate synthase (EC: 4.1.1.48, InGPS) encoded by the trpC gene. The fifth enzyme involved in the biosynthesis of tryptophan is tryptophan synthase (EC: 4.2.1.20, TS) encoded by the trpB and trpA genes. This enzyme catalyzes the final two steps in the biosynthesis of tryptophan. The product of trpA catalyzes the reversible cleavage of indole-3-glycerol-phosphate to form indole and glyceraldehyde 3-phosphate, whereas the product of trpB catalyzes the irreversible condensation of indole and serine to form tryptophan [1]. ∗
The structural organization of the third enzyme, phosphoribosyl anthranilate isomerase, of the tryptophan synthesis pathway differs widely among microorganisms. It is a monofunctional enzyme of a single polypeptide chain in Acinetobacter calcoaceticus [2], Bacillus subtilis [3], Pseudomonas putida [4], and Saccharomyces cerevisiae [5]. However, in Aerobacter aerogenes [6], Escherichia coli [7], Salmonella typhimurium [8], and Serratia marcescens [9], it is found together with indole-3-glycerol-phosphate synthase in the same polypeptide chain. This enzyme has been characterized from all the three domains of life including eukaryotes [10–12], archaea [13–15], and bacteria [16–18]. Phosphoribosyl anthranilate isomerase has been characterized from several bacteria however, to the best of our knowledge, there is no report available from the genus Geobacillus. We are interested in the tryptophan metabolism in thermophilic microorganisms. We have previously characterized anthranilate phosphoribosyltransferase and phosphoribosyl anthranilate isomerase from the hyperthermophilic archaeon Thermococcus kodakarensis [15,19], and indole-3-glycerol-phosphate synthase from the hyperthermophilic archaeon Pyrococcus furiosus [20]. Recent ease in genome sequencing has piled up the amino acid sequences of uncharacterized enzymes including those involved in tryptophan synthesis. The genome sequence of Geobacillus thermopakistaniensis, a thermophilic bacterium, has been determined [21]. The genome sequence analysis revealed the presence of a phosphoribosyl anthranilate isomerase homologue; however, the
Corresponding author. E-mail addresses: [email protected], [email protected] (N. Rashid).
https://doi.org/10.1016/j.pep.2019.105523 Received 26 August 2019; Received in revised form 18 October 2019; Accepted 23 October 2019 Available online 29 October 2019 1046-5928/ © 2019 Elsevier Inc. All rights reserved.
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enzyme has not been functionally characterized yet. In this manuscript we describe molecular cloning and characterization of this enzyme as a first step of understanding of the tryptophan metabolism in G. thermopakistaniensis.
against their retention volumes. The standard proteins used were lysozyme (14 kDa), proteinase K (29 kDa), ovalbumin (44 kDa), bovine serum albumin (66 kDa), and alcohol dehydrogenase (147 kDa). 2.5. Activity assay
2. Materials and methods
Enzyme activity of TrpFGt was determined by a coupled enzyme assay system by using the thermostable TrpD from T. kodakarensis (prepared in our lab) as shown in the scheme below.
2.1. Reagents, vectors and bacterial strains The reagents used in this study were of analytical grade and purchased either from Thermo Fisher Scientific, USA or Sigma-Aldrich, USA or Fluka Chemicals, UK. Restriction enzymes and DNA purification kits were purchased from Thermo Fisher Scientific. The gene specific primers were chemically synthesized from Macrogen, Korea and expression vector pET-21a was purchased by Novagen®, USA. The genetically engineered E. coli strains DH5α and BL21- CodonPlus (DE3)RIL were procured from Stratagene, USA.
Anthranilate + Phosphoribosyl pyrophosphate (PRPP ) anthranilate (PRA) + pyrophosphate (PPi) Phosphoribosyl anthranilate (PRA) 1
TrpFGt
1
(o
deoxyribulose 5
Phosphoribosyl
carboxphenyla min o) phosphate (CDRP )
As the substrate for TrpFGt is highly unstable at higher temperatures therefore a continuous coupled enzyme activity assay was performed. For the preparation of the substrate, the initial assay mixture, 1.95 mL, containing 0.1 M Tris-HCl (pH 8.0), 33 μM anthranilate, 100 μM ZnCl2 and 10 μg of TkTrpD (an anthranilate phosphoribosyl transferase from T. kodakarensis) was incubated at 60 °C (optimal temperature for TkTrpD) for 5 min. The reaction was initiated with the addition of phosphoribosyl pyrophosphate (1 mM) and 10 μg of TrpFGt. After 3 min of incubation at 60 °C, the reaction was stopped by quenching in ice water. The decrease in the amount of anthranilate was measured with a fluorescent spectrophotometer. The activation and emission wavelengths were set at 310 nm and 390 nm, respectively. By using a standard curve, the amount of carboxyphenylamino-deoxyribulose 5-phosphate produced per min was calculated (production of 1 μmol of carboxyphenylamino-deoxribulose 5-phosphate is equivalent to consumption of 1 μmol of anthranilate).
2.2. Cloning and expression constructs The gene encoding phosphoribosyl anthranilate isomerase from G. thermopakistaniensis, trpFGt (accession # ESU71245) was amplified by polymerase chain reaction (PCR) by utilizing genomic DNA of G. thermopakistaniensis as template and commercially synthesized forward ( 5′-CATATGATTCGAATTAAATATTGCGGCAACCG) and reverse (5′-TCA TCGACACCCGTGATGTT) primers. An NdeI site (underlined sequence) was introduced in the forward primer. The PCR amplified DNA fragment was ligated in pTZ57 R/T cloning vector. The resulting recombinant vector was designated as pTZ-TrpFGt. The TrpFGt gene was excised from pTZ-TrpFGt using NdeI (incorporated in the forward primer) and BamHI (present in multiple cloning site of pTZ57 R/T). The excised gene was sub-cloned in pET-21a expression vector utilizing the same restriction sites. The resulting recombinant plasmid was denoted as pET-TrpFGt.
2.6. Effect of chemical denaturants The effect of chemical denaturants on the stability of TrpFGt was examined by incubating the protein samples with various final concentrations of urea (0–8 M) and guanidinium hydrochloride (0–6 M) for 45 min at room temperature. The residual activities of the samples were examined as described above. In addition to the residual activities, these samples were analyzed by circular dichroism (CD) spectroscopy using a Chirascan™-plus CD Spectrometer (Applied Photophysics). CD spectra were recorded in the far-UV region (200–280 nm).
2.3. Production and purification of TrpFGt The recombinant plasmid, pET-TrpFGt, was used to transform E. coli BL21-CodonPlus (DE3)-RIL cells and cultivated at 37 °C in Luria–Bertani broth containing 100 μg/mL ampicillin till the optical density, at 660 nm, reached to 0.4–0.6. The gene expression was induced by the addition of 0.2 mM isopropyl-β-d-1- thiogalactopyranoside for 5–6 h. The cell pellet (~3 g wet weight from 1 L culture), obtained by centrifugation, was resuspended in 50 mM Tris-HCl buffer (pH 8) and lysed by sonication. Soluble and insoluble fractions were separated by centrifugation at 20,000 × g. The soluble fraction was subjected to heat-treatment at 60 °C for 25 min and centrifuged at 20,000 × g to remove heat-denatured proteins of E. coli. ÄKTA Purifier system (GE Healthcare) was employed for further purification of recombinant TrpFGt using a HiTrap® Q anion exchange column (GE Healthcare). Elution of the proteins bound to the column was performed by using a linear gradient of 0–1 M NaCl. The fractions containing a significant amount of TrpFGt were pooled, dialyzed against 50 mM Tris-HCl (pH 8.0) and applied to Resource™ Q, another anion exchange column (GE Healthcare). The bound proteins were eluted in a similar way using a linear gradient of 0–1 M NaCl. Quantification of purified TrpFGt was done spectrophotometrically using Bradford reagent (BioRad) and purity of the protein was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
2.7. Structural modeling The three-dimensional structural model of TrpFGt was obtained from I-TASSER [22]. The model obtained was evaluated by its C-score. The structure obtained was compared with the previously reported crystal structures of phosphoribosyl anthranilate isomerase from Thermotoga maritima (1NSJ) and E. coli (1PII) using PyMOL software (https:// pymol.org/). 3. Results The gene, trpFGt, encoding phosphoribosyl anthranilate isomerase in G. thermopakistaniensis comprises 642 nucleotides corresponding to a polypeptide of 213 amino acids. The calculated molecular weight and isoelectric point of the protein were 23,090 Da and 6.06, respectively. A homology search demonstrated that TrpFGt was 100% identical in primary structure with an uncharacterized phosphoribosylanthranilate isomerase from Geobacillus zalihae (accession # WP_081132668). Among the characterized enzymes, highest identity of 48.5% was found with the counterpart from Bacillus subtilis (WP_004429772), followed by 35% from Pseudomonas putida (WP_065861173) and 32.5% from E. coli (AIT70912). A comparison of sequences of the characterized
2.4. Gel filtration chromatography Gel filtration chromatography was performed by using a Superdex 200 Increased 10/300 column (GE Healthcare) equilibrated with 100 mM NaCl in 10 mM Tris–HCl pH 7.5. The standard curve was obtained by plotting the log of molecular weights of standard proteins 2
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Fig. 1. Alignment of six conserved regions found in the characterized TrpF enzymes from bacteria and archaea. The amino acid residues involved in substrate binding and catalysis in TrpF from T. kodakarensis and T. thermophilus are shown in bold and bold-underlined, respectively. Names at the left-hand side indicate the source organism from which the sequence originated. The numbers show the position of the amino acid in the protein sequence. The accession numbers of the sequences are: B. subtilis (BSU22650), Chlamydia trachomatis (B0BBV9), E. coli (AGW08587), Haloferax volcanii (P52563), T. thermophilus (YP_143342), T. maritima (AAD35232), Acinetobacter calcoaceticus (AQZ83080), Pseudomonas putida (Q88LE0), T. kodakarensis (Q9YGB1) and P. furiosus (Q8U092).
phosphoribosyl anthranilate isomerases from bacteria and archaea (Fig. 1) demonstrated that thermostable enzymes contain a higher number of Glu and Lys residues, and a lower percentage of Gln and His in agreement with the literature [20,23]. From the sequence alignment, six highly conserved regions found in bacterial and archaeal enzymes were figured out (Fig. 1). The amino acid residues involved in catalysis and substrate binding in the enzyme from T. kodakarensis [15], completely conserved in the characterized enzymes from bacteria and archaea, are shown Fig. 1.
3.3. Effect of metal ions Addition of 1 mM EDTA in the assay mixture drastically decreased the activity which indicated that TrpFGt activity might be dependent on metal ions. Therefore, we examined the effect of various metal ions at a final concentration of 100 μM. A two-fold enhancement in the activity was found in the presence Zn+2 (Fig. 4). Presence of Ca+2 and Mg+2 also enhanced the activity but not significantly. Enhancement of activity in the presence of Zn+2 is a unique feature of TrpF. All the previously characterized phosphoribosyl anthranilate isomerases are reported to be activated in the presence of Mg+2.
3.1. Biosynthesis and purification of TrpFGt
3.4. Structural stability against temperature and denaturants
Induction of host E. coli cells carrying pET-TrpGt plasmid resulted in production of recombinant protein in soluble and active form at high levels, almost 35% of E. coli proteins. Heat-treatment, the initial purification step, removed most of the heat-labile proteins of the host. Ionexchange chromatography of the soluble part, after heat treatment, resulted in an apparent homogeneous protein band on SDS-PAGE (Fig. 2A). Various steps involved in purification of recombinant TrpFGt and respective enzyme activities at each step are summarized in Table 1. When analyzed by gel filtration, TrpFGt eluted at a retention volume of 16.9 mL, equivalent to a molecular weight of ~50 kDa (Fig. 2B). This result indicated that recombinant TrpFGt existed in a dimeric form in solution, similar to the thermostable counterparts from T. maritima [25] and T. thermophilus [18].
As the optimal temperature for TrpFGT activity was 60 °C, we therefore measured the stability of the enzyme at this temperature. TrpFGT was quite stable at this temperature with a half-life of 90 min (Fig. 5A). However, at higher than 60 °C, the activity reduced drastically. We further examined the effect of denaturants by incubating TrpFGt, at room temperature for 45 min, in the presence of 0–8 M urea or 0–6 M Gdn-HCl (final concentration) and measuring the residual activity. There was no detectable effect even at 8 M urea. However, the activity gradually decreased with the increasing concentration of GdnHCl. There was more than 40% decrease in activity even at 2 M. The activity was completely inhibited at 6 M (Fig. 5B). Apart from activity measurements, these protein samples were analyzed by CD spectrometry. There was no significant change in CD spectra of the samples incubated in the presence or absence of various concentrations of urea (Fig. 5C). However, there were significant changes in CD spectra of TrpFGt samples incubated in the presence of Gdn-HCl (Fig. 5D), which matched the residual activities of these samples.
3.2. Optimal temperature and pH When the reaction catalyzed by TrpFGt was carried out at various temperatures (20–80 °C), the enzyme activity increased gradually with the increase in temperature till the highest at 60 °C (Fig. 3A). Similarly, when we measured the activity by varying the pH from 5.5 to 9.5 and keeping the temperature unchanged at 60 °C, the highest activity was found at pH 8.0 (Fig. 3B).
3.5. Kinetic parameters Measurements of kinetic parameters were performed by plotting the initial reaction velocities against various substrate concentrations. The reaction catalyzed by TrpFGt followed Michaelis-Menten kinetics 3
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Fig. 3. Effect of temperature and pH on enzyme activity of TrpFGt. A) Effect of temperature. Activity assays were performed at various temperatures ranging from 30 to 80 °C at pH 8.0 in 50 mM Tris-HCl. B) Effect of pH. Activity assays were performed at 60 °C and various pH. Two buffer systems, Na-phosphate buffer pH 6.0–7.5 (squares) and Tris-HCl buffer pH 7.5–9.5 (circles), were used. Error bars indicate standard deviation.
Fig. 2. Purity and molecular weight determination. A) Coomassie brilliant blue stained 15% SDS-PAGE showing the purified recombinant TrpFGt. Lane M, molecular weight marker; lane 1, purified TrpFGt (10 μg). B) Plot of log of molecular weight of the standards used for gel filtration chromatography against Kav, calculated from their retention volumes, for molecular weight and subunit determination. Filled circles show the position of molecular weight standards while the open circle shows the position of TrpFGt. Error bars indicate standard deviation. Table 1 Purification of recombinant TrpFGt. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification-fold
Yield (%)
Total cell lysate Heat treatment HiTrap QFF Resource Q
60 17 11 8
5023 4196 3677 3176
84 247 334 397
1 3 4 4.7
100 84 73 63
Fig. 4. Effect of various metal cations on TrpFGt activity. Reactions were performed at 60 °C and pH 8.0. Chloride salt of each metal cation was used at a final concentration of 100 μM. EDTA, when used, was 1 mM. Each measurement is the average value of three independent experiments.
4
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Fig. 5. Structural stability of TrpFGt against temperature and denaturants. A) Thermostability of TrpFGt. The protein was incubated at 60 °C for various time intervals and residual activity was measured at 60 °C and pH 8.0 in Tris-HCl buffer. B) Effect of denaturants. TrpFGt was incubated at room temperature for 45 min in the presence of various concentrations of urea (circles) and guanidine-HCl (squares) and residual activity was measured. C) Structural stability against urea. Before measuring the CD spectra, TrpFGt was incubated at room temperature for 45 min in the presence of 0 (open squares), 2 (filled squares), 4 (filled triangles), and 8 M (open circles) urea (final concentration). D) Structural stability against guanidinium-HCl. Before measuring the CD spectra, TrpFGt was incubated at room temperature for 45 min in the presence of 0 (squares), 4 (circles) and 6 M (triangles) guanidinium-HCl. CD spectra were recorded in the far-UV region (200–280 nm).
(Fig. 6A). The apparent Vmax and Km values were 480 ± 10 μmol min−1 mg−1 and 1.15 ± 0.05 μM, respectively. The Vmax exhibited by TrpFGt is the highest among the characterized phosphoribosyl anthranilate isomerases. TrpFGt exhibited a kcat value of 185 s−1 subunit−1. The low Km and the high kcat values of TrpGGt, compared to its bacterial counterparts, indicate that catalytically it is a highly efficient enzyme. The reaction catalyzed by TrpFGt at various temperatures followed a linear trend till 60 °C. By using the rates of reaction at various temperatures, an Arrhenius plot (lnk vs. 1/T) was used to calculate the activation energy which came out to be 9.02 ± 0.5 kJ mol−1 (Fig. 6B). Activation enthalpy and entropy calculated from Eyring–Polanyi plot (lnk/T vs. 1/T) were and –116 ± 1 J mol−1 K−1, respectively 16.5 ± 0.5 kJ mol−1 (Fig. 6C).
substrate binding site. 4. Discussion Recent advancements have in DNA sequencing has piled up the amino acid sequences of the uncharacterized proteins. A protein data base search provided by National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/protein/?term=phosphoribosyl anthranilate + isomerase) revealed more than ninety thousand protein sequences annotated as phosphoribosylanthranilate isomerases. However, a very few of them have been experimentally verified to be true phosphoribosylanthranilate isomerase. Experimental examination of these uncharacterized proteins is expected to lead the discovery of novel enzymes. We have previously characterized a few enzymes involved in tryptophan metabolism in T. kodakarensis [15,19] and P. furiosus [20]. We have described here, TrpFGt, a phosphoribosylanthranilate isomerase from G. thermopakistaniensis, which was produced in E. coli in soluble and highly active form. A novel feature of TrpFGt was its highest activity, 480 μmol min−1 mg−1, among the characterized counterparts. Similarly, the kcat/Km exhibited by TrpFGt is the highest among the characterized phosphoribosylanthranilate isomerases, indicating that it is a highly efficient enzyme. TrpFGt exhibited highest activity in the presence of Zn+2 whereas the enzyme from E. coli exhibited highest activity in the presence of Mg+2. TrpFGt is the only
3.6. Structural analysis The structural model obtained from I-TASSER had C-score of 1.25, signifying high confidence of the predicted model. TrpFGt structural model was superimposed with the previously reported crystal structures of its counterparts from T. maritima and E. coli (Fig. 7). The TrpFGt structure shows more similarity with the one from T. maritima (RMSD of 0.75) as compared to that of E. coli (RMSD of 1.20). However, it has notable differences from the other two structures, particularly near the 5
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Fig. 7. Superimposition of the three-dimensional structure of TrpFGt (red) on the crystal structures of phosphoribosyl anthranilate isomerases from T. marimtima (blue) and E. coli (green). The residues involved in substrate binding and catalysis are labeled and shown as sticks. The figure was drawn in PyMOL (https://pymol.org/).
proteins from hyperthermophilic archaea and thermophilic bacteria are reported to maintain their native structures, and hence the enzyme activities, in the presence of these denaturants [26-28]. We also examine the activity of TrpFGt after incubating with various concentrations of urea or guanidinium chloride and found that there was no significant loss in activity even after incubating in 8 M urea. However, the activity was reduced to 20% in the presence of 6 M guanidinium chloride. This could be due to the fact that guanidinium chloride is a salt as well as a denaturant whereas urea is an uncharged molecule, hence, has less ionic strength effects. The predicted model of TrpFGt indicates that the binding site has structural differences with its counterparts from T. maritima and E. coli that may account for its unique characteristics. In conclusion, the results obtained in this study demonstrate that TrpFGt is highly resistant to urea, a protein denaturant. It exhibits the highest ever reported phosphoribosyl anthranilate isomerase activity. Another unique feature of the enzyme is its activation by Zn+2. Further studies are underway to elucidate the structure function relationship of the enzyme. Declaration of competing interest The authors declare no conflict of interest.
Fig. 6. Determination of kinetic parameters. A) Substrate-dependent enzyme activity of TrpFGt. The data was used to fit the Michaelis–Menten equation to obtain the apparent Km and Vmax values. B) Arrhenius plot (lnk versus reciprocal absolute temperature) for the activation energy of TrpFGt. C) Eyring–Polanyi plot (ln(k/T) versus reciprocal absolute temperature) for calculation of enthalpy and entropy of the reaction catalyzed by TrpFGt.
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