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Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1
ARTICLE IN PRESS Microbiological Research 160 (2005) 53—59
www.elsevier.de/micres
Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 D.Q. Liua,b,c, H. Liua, X.L. Gaoa, D.J. Leakd, N.Y. Zhoua, a
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China c Graduate School of Chinese Academy of Sciences, Beijing 100039, China d Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK b
Accepted 24 September 2004
KEYWORDS 3-hydroxybenzoate 6-hydroxylase; Site-directed mutagenesis; Klebsiella pneumoniae M5a1
Summary 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis. & 2004 Elsevier GmbH. All rights reserved.
Introduction Klebsiella pneumoniae M5a1, a soil bacterium, used extensively in molecular genetic studies of dinitrogen fixation (Dixon, 1984) and various kinds of genetic manipulation, was found to be able to degrade many aromatic compounds including some hydroxybenzoates. The genes encoding the enzymes that convert 3-hydroxybenzoate (3-HBA) to pyruvate and fumarate via the gentisate pathway
were located on an 8 kb SphI fragment of K. pneumoniae M5a1 DNA (Robson et al., 1996). This fragment was sequenced in both directions, revealing seven complete open reading frames (Gao, 2003). By cloning, expression and biochemical analysis of the fragment, four of these genes (mhbMDIH) were shown to encode the enzymes involved in the catabolism of 3-HBA to fumarate and pyruvate through the gentisate pathway, of which MhbM is a 3-HBA 6-hydroxylase that catalyzes
Abbreviations: 3-HBA, 3-hydroxybenzoate; MhbM, 3-hydroxybenzoate 6-hydroxylase encoded by mhbM from Klebsiella pneumoniae M5a1; IPTG, isopropyl-b-D-thiogalactopyranoside Corresponding author. Tel./fax: 86 27 87197655. E-mail address: [email protected] (N.Y. Zhou). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.09.003
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the para-hydroxylation of 3-HBA to 2, 5-dihydroxybenzoate (gentisate), consisting of 397 amino acids with a predicted mass of 43.9 kDa (Gao, 2003). 3-HBA 6-hydroxylase (or 3-HBA 6-monooxygenase) [EC 1.14.13.24] from K. pneumoniae M5a1 is a member of the family of NAD (P) H-dependent flavoprotein hydroxylases (Sua ´rez et al., 1995). Unlike other hydroxylases which catalyze orthohydroxylation, 3-HBA 6-hydroxylase introduces a new hydroxyl group at the para-position to the existing one and yields gentisate (2, 5-dihydroxybenzoate) (Fig. 1), an intermediate step in the degradation of aromatic compounds in soil bacteria (Groseclose et al., 1973; Wang et al., 1987; Yu et al., 1987; Rajasekharan et al., 1990; Sua ´rez et al., 1995). In contrast to the ortho-hydroxylation, few studies were carried out about the para-hydroxylation during the last two decades (Zhou et al., 2001). However, gentisate and substituted gentisates are key intermediates in the aerobic pathway for the metabolism of a large number of aromatic compounds, including 3-hydroxybenzoate (Jones and Cooper, 1990; Goetz and Harmuth, 1992), substituted phenols (Crawford and Frick, 1977;
Poh and Bayly, 1980; Jain, 1996), naphthalene (Monticello et al., 1985; Grund et al., 1992; Fuenmayor et al., 1998), salicylate (Ohmoto et al., 1991; Rani et al., 1996), and 3, 6-dichloro-2methoxybenzoate (Werwath et al., 1998). Till now, 3-hydroxybenzoate 6-hydroxylase has been purified from K. pneumoniae M5a1 (Sua ´rez et al., 1995), Pseudomonas cepacia (Wang et al., 1987; Yu et al., 1987), Micrococcus sp. (Rajasekharan et al., 1990), and P. aeruginosa (Groseclose et al., 1973). Their properties were summarized in Table 1. However, no DNA sequences available so far except the mhbM from K. pneumoniae M5a1, which has also been functionally expressed. As little is known about the mechanism of parahydroxylation about this enzyme, it will be interesting to define the amino acid residues which are involved in catalytic activity of 3-HBA 6-hydroxylase. However, the three-dimension crystal structure of this enzyme is not available yet. To identify the residue which is critical for catalysis, sitedirected mutagenesis was carried out to replace Arg with Glu at position 169 in the strictly conserved motif among related enzymes that were alighted. The effects of the mutant on the activity
3-hydroxybenzoate 6-hydroxylase MhbM COOH
O2
COOH
H2O
OH
OH
NAD(P)H + H+ NAD(P) +
3-hydroxybenzoate
HO Gentisate
Figure 1. 3-hydroxybenzoate 6-hydroxylase catalyzes para-hydroxylation of 3-hydroxybenzoate to form gentisate in K. pneumoniae M5a1.
Table 1.
Comparison of 3-hydroxybenzoate 6-hydroxylases (or monooxygenases) from different strains
Enzyme
Strain
Mw(kDa)
Electron donor
Subunits
Prosthetic group
Optimal pH
3-hydroxybenzoate 6-monooxygenase 3-hydroxybenzoate 6-monooxygenase
P. cepacia
44.0
Monomer
FAD
8.0
P. aeruginosa
85.0
Monomer
FAD
8.0
3-hydroxybenzoate 6-monooxygenase
Micrococcus sp.
85.0
Monomer
FAD
8.2
3-hydroxybenzoate 6-monooxygenase
K. pneumoniae M5a1
42.0
NADH or NADPH NADH, preferred to NADPH NADH, preferred to NADPH NADH or NADPH
Monomer
FAD
8.0
ARTICLE IN PRESS Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1
of this enzyme were analyzed by color and spectrophotometric assays.
Materials and methods Strains, plasmids and general methods Escherichia coli Rosetta (DE3) pLysS (Novagen, USA) were used as the recipient strains for recombinant plasmids, which contains tRNAs for rare codons in E. coli Rosetta (DE3) pLysS. Rosetta cells were grown in LB medium at 37 1C; Kanamycin and chloramphenicol were added to final concentrations at 60 and 34 mg/ml, respectively for plasmid selection. IPTG and 3-HBA were obtained from Promega and Sigma, respectively. PyrobestTM DNA Polymerase (high fidelity), restriction and ligation enzymes were supplied by Takara Company (Dalian, China), and used as recommended by the manufacturers. DNA preparation of genes cloned into pET28a(+) (Novagen, USA), agarose gel electrophoresis, restriction enzyme digestion, DNA ligation and transformation were done by standard procedures (Sambrook et al., 1989). Purification of PCR products and DNA fragments from agarose gel was done by using the PCR purification kit from Xinzhi Biotechnology, Ltd. (Shanghai, China). Nucleotide sequences were determined by United Gene Holdings, Ltd. (Shanghai, China)
Site-directed mutagenesis The mhbM gene encoding the 3-HBA 6-hydroxylase from K. pneumoniae M5a1 was amplified from the 8 kb SphI fragment of K. pneumoniae M5a1 DNA (Robson et al., 1996) by PCR using PyrobestTM DNA Polymerase. The following oligonucleotide primers were used for this purpose: (1) 50 GCTTAGGCATTCATATGGCAAAAGTGAT30 , and (2) 50 AGGCTACGAATTCACCGCTATCTGC30 . The NdeI and EcoRI site were introduced into the primers (underlined), respectively in primers 1 and 2. The amplified PCR product was then excised with NdeI and EcoRI, ligated into the expression vector pET28a(+), and transformed into E. coli DH5a to produce plasmid pZWGM28. Overlap extension PCR was performed to introduce mutations in mhbM using the method described by Pogulis et al. (1996). Primers used to create the mutant R169E were designed as follows: (3) 50 GGTGGTGGAGCAAAGTCTGCTGG30 , and (4) 0 5 CAGACTTTGCTCCACCACCGACTTAC30 . The nucleotides mutated are underlined. The inserts of the
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mhbM wild-type and its mutant’s clones were sequenced in both directions to ensure no mutation (for pZWGM28) or unintended mutation (for R169E) had been incorporated during the PCR prior to expression and characterization studies.
Expression of wild-type and mutant enzymes in Rosetta Rosetta cells carrying pET28a(+) containing either the wild-type or mutant insert were grown at 37 1C with shaking in LB broth with Kanamycin and chloramphenicol. When the OD600 reached 0.6, IPTG was added to final concentration of 1 mM and incubation continued for 4 h before the cells were harvested by centrifugation. Cell extracts were prepared by resuspending the bacterial pellets in ice-cold 25 mM phosphate buffer (pH 7.5) and disruption by sonication in an ice-water bath for 30 min (5 s burst per 2 s cooling), after which cell debris was removed by centrifugation at 15,000g for 30 min at 4 1C.
Enzyme assay The 3-HBA 6-hydroxylase activity assay was performed at 30 1C by measuring the rate of decrease in absorbance at 340 nm due to the substratedependent oxidation of NADH (Wang et al., 1987; Zhou et al., 2002). The sample cuvette contained 50 mM potassium phosphate buffer (pH 8.0), 0.05 mmol 3-HBA and 0.15 mmol NADH in 1-ml volume. The reference cuvette contained the same except the substrate. The assay was initiated by adding the component of 3-HBA 6-hydroxylase to both cuvettes. The specific activity of 3-HBA 6hydroxylase was calculated from the initial DA340/ min, taking the molar extinction coefficient at 340 nm for NADH as 6220 M1 cm1. Protein concentrations were determined by the Bradford method (Bradford, 1976). Specific activities are expressed as units per milligram of protein. SDSPAGE was performed according to the manufacture’s instructions (Bio-Rad). Qualitative assay was also carried out by color assay. In this assay, 3-HBA, IPTG, Kanamycin, and chloramphenicol were added to LB agar plates with final concentrations of 2 mM, 1 mM, 60 mg/ml, and 34 mg/ml, respectively. Rosetta cells carrying wildtype mhbM or its mutant clone were streaked on above plates and incubated at 30 1C overnight. An accumulation of the brown oxidation products of gentisate (Fuenmayor et al., 1998) will be observed when a clone carrying a functional mhbM gene that
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catalyzes the para-hydroxylation of colorless 3HBA.
Biotransformation Biotransformation (modified from Zhou et al., 2002) of 3-HBA was performed by growing Rosetta cells carrying wild-type mhbM and mutant R169E in 200 ml of LB medium containing 2 mM substrate. Kanamycin(60 mg/ml), chloramphenicol (34 mg/ml), and IPTG (1 mM) were also present in the medium. The cultures were incubated with shaking at 150 rpm and 30 1C. The cleared samples were checked periodically until the spectra of the sample had no change (between 24 and 36 h).
Computer analysis of protein sequence Nucleotide and amino acid sequence similarities were determined by BLAST-N and PSI-BLAST Searches (Altschul et al., 1990, 1997). Protein motifs were identified by BLAST-P (Altschul et al., 1997) and BLOCKs (Henikoff and Henikoff, 1994). Secondary structure prediction and analysis were performed by PHD (Rost, 1996) and TMHMM (Sonnhammer et al., 1998). Multiple sequence alignment was assembled by CLUSTAL-W (Thompson et al., 1994).
Nucleotide sequence accession number The DNA sequence of 1194 bp of the complete mhbM gene has been submitted to GenBank under accession number AY648560.
Results Constructing of mutant R169E of mhbM By amino acids sequence multiple alignments with several closely related enzymes, a high conservation of the serine and arginine was found at positions 166 and 169 of 3-HBA 6-hydroxylase from K. pneumoniae M5a1 (as shown in Fig. 2). The block K. pneumoniae M5a1 P. putida S-1s P. putida P. stutzeri Sphingomonas sp.
searcher and motif analysis showed Ser166 and Arg169 formed the highly conservative motif SXXR, which was putatively to belong to the monooxygenase family which utilizes FAD. The strictly conserved amino acids of this motif were expected to play an important role in the structure and function of the enzyme. It has been reported that an arginine residue participates the binding of 3-HBA to 3-HBA 6hydroxylase from Micrococcus sp, although it did not define which Arg was involved (Sumathi et al., 1998); A study on salicylate hydroxylase from P. putida (EC 1.14.13.1), an FAD-containing monooxygenase, also suggested that one arginine residue of the enzyme is responsible for the NADH binding site (Suzuki and Ohnishi, 1990). Therefore, sitedirected mutagenesis was carried out to obtain the mutant mhbM gene containing the aimed substitution of amino acid at position 169, from arginine to glutamate. The overlap PCR was performed using primers as described in section Materials and methods. Wild-type and mutated fragments which encoding the enzyme was inserted to T7 promoter directed plasmid pET28a(+). DNA sequencing confirmed that no mutation had been incorporated during the PCR for mhbM wild-type clone but there were indeed desired mutations created in mutant R169E (CGC to GAG).
Expressions of the wild-type and mutant enzymes and activity assays Expressions of the wild-type and mutant enzymes were performed at 37 and 30 1C. SDS-PAGE of the cell extracts (Fig. 3) showed elevated levels of a polypeptide of 44 kDa from both wild-type and mutant R169E, as expected from the deduced amino acid composition of MhbM. They are also predominantly present in the soluble form from the cell induced at 30 1C (data not shown). However, spectrophotometric assay showed that R169E mutant had completely lost enzyme activity, but the wild-type enzyme displayed 3-HBA-6-hydroxylase at a specific activity of 0.53 U/mg under the same condition.
DDVTV-FDDKGNSWTADILIGCDGGKSVVRQSLLGDSPRV------TGHV DELQVLFRD-GTEYRCDLLIGRDGIKSALRSYVLEGQGQDHLEPRFSGTC EGVTLNFAD-GSTYTADVAIAADGIKSSMRNTLLRAAGHDAVHPQFTGTS EQVVLHFKDGSKA-EADIVIGADGMRSTVRNLMLGYEDYI-----YAGYT DGVELRFAGRDPV-IADVVIGADGVRSVIRDFVTGGRAAL-----YSGTS
183 191 187 160 179
Figure 2. Multiple amino acids sequence alignment of the MhbM and its related enzymes. Aligned sequences are 3hydroxybenzoate 6-hydroxylase from K. pneumoniae M5a1; Salicylate hydroxylase from P. putida S-1 s (accession number BAA61829); Salicylate hydroxylase from P. putida (accession number Q53552); Salicylate hydroxylase from P. stutzeri (accession number AAD02157); Salicylate hydroxylase from Sphingomonas sp (accession number BAA19150). Conserved residues are shaded in black.
ARTICLE IN PRESS Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1
Figure 3. SDS-PAGE of overexpressed 3-HBA 6-hydoxylase (MhbM) and its mutant R169E in E. coli on a 12% gel. Lane 1, cell extracts containing MhbM obtained after induction with IPTG; lane 2, marker; lane 3, pET28a(+) alone; lane 4, E. coli Rosetta (DE3) pLysS alone; lane 5, cell extracts containing mutant R169E obtained after induction with IPTG. Marker masses are indicated on the left in kilodaltons. The molecular mass of the overexpressed protein (indicated by the large arrow on the right) is 44 kDa.
Qualitative color assay also showed the media surrounding the cell carrying the wild-type mhbM had changed to a brown color, indicating presence of a functional 3-HBA 6-hydroxylase; whereas no accumulation of the brown color on the plates containing the mutant, implying the mutant being no longer able to convert 3-HBA to gentisate. In the biotransformation studies, after 24 h the E. coli cells carrying wild-type 3-HBA 6-hydroxylase converted 3-HBA with a maximum wavelength (lmax) of 290 nm to a product with lmax of 320 nm, a characteristic of gentisate (Fuenmayor et al., 1998). But the mutant R169E could not transform 3HBA to the product formed from the wild-type enzyme and the spectrum of gentisate in the culture remained the same during the course.
Discussion 3-HBA 6-hydroxylase is an important enzyme in the gentisate pathway of 3-HBA degradation by catalyzing para-hydroxylation. Another example catalyzing para-hydroxylation in gentisate pathway is a salicylate 5-hydroxylase from Ralstonia sp. strain U2, which converts salicylate to gentisate. But it is
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a three-component system (a/b oxygenase components, ferredoxin reductase and ferredoxin) (Zhou et al., 2002) rather than being in the singlecomponent flavoprotein hydroxylases family, which 3-HBA 6-hydroxylase from K. pneumoniae M5a1 belongs to. Based on the presence of a highly conservative motif in MhbM and related FADcontaining monooxygenases, and evidences that an arginine is important for the activities of these enzymes for the enzymes’ activity, Arg169 was chosen for site-directed mutagenesis studies. Three independent enzyme activity assays clearly suggested that the substitution of arginine to glutamate at position 169 in the 3-HBA 6hydroxylase had resulted in the complete loss of its catalytic activity. This indicates Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis. After the Arg residue, a basic amino acid, substituted with Glu, a negative charged amino acid, both the charged character and ionized state of the residue at position 169 may lead to the failure of forming salt bond between 3-HBA and 3HBA 6-hydroxylase. Previous chemical modification investigations have suggested that an arginine residue participates the binding of 3-HBA to 3HBA 6-hydroxylase (from Micrococcus sp.) through electrostatic interaction (Sumathi et al., 1998), but it did not define which Arg was involved, apparently due to lack of sequence information. From the evidence described previously, Arg169 in MhbM may be a very important residue that participates the binding of 3-HBA to the enzyme. Another study on salicylate hydroxylase (converting salicylate to catechol) from P. putida, one of related enzymes to 3-HBA 6-hydroxylase, showed that an arginine residue at the active site of this enzyme not only acts as an NADH binding site, but also plays an essential role in maintaining a conformational state for the oxygenation of enzyme-substrate (Suzuki and Ohnishi, 1990). As to the Arg169 of 3-HBA 6hydroxylase, more experiments are needed to determine whether it is also involved in the NADH binding site. Secondary structure analysis showed no conformational transformation of MhbM was induced, after Arg was changed to Glu at position 169. The loss of enzyme activity was indicative of the importance of Arg169 in the functionality of 3-HBA 6-hydroxylase. However, when a highly conserved residue Arg 166 (equivalent to Arg169 in MhbM) was substituted with Glu in 4-hydroxybenzote hydroxylase (converting 4-hydroxybenzote to 3, 4-dihydroxybenzote) from P. fluorescens, it was found to have caused structural perturbations, and impaired binding of NADPH and FAD (Eppink et al., 1999). A further study on the structure analysis of MhbM will
ARTICLE IN PRESS 58 then be necessary to clarify the function of this or other residues essential to this important enzyme.
Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 30170036) and by the ‘‘Hundred Talents’’ program from Chinese Academy of Sciences to NYZ. We also thank Mrs. S. J. Wang for her technical assistance.
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D.Q. Liu et al. Henikoff, S., Henikoff, J.G., 1994. Protein family classification based on searching a database of blocks. Genomics 19, 97–107. Jain, R.K., 1996. The molecular cloning of genes specifying some enzymes of the 3, 5-xylenol degradative pathway. Appl. Microbiol. Biotechnol. 45, 502–508. Jones, D.C.N., Cooper, R.A., 1990. Catabolism of 3hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5a1. Arch. Microbiol. Biotechnol. 154, 489–495. Monticello, D.J., Bakker, D., Schell, M., Finnerty, W.R., 1985. Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate. Appl. Environ. Microbiol. 49, 761–764. Ohmoto, T., Sakai, K., Hamada, N., Ohe, T., 1991. Salicylic acid metabolism through a gentisate pathway by pseudomonas sp TA-2. Agric. Biol. Chem. 55, 1733–1737. Pogulis, R.J., Vallejo, A.N., Pease, L.R., 1996. In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol. Biol. 57, 167–176. Poh, C.L., Bayly, R., 1980. Evidence for isofunctional enzymes used in m-cresol and 2, 5-xylenol degradation via gentisate pathway in pseudomonas alcaligenes. J. Bacteriol. 143, 59–69. Rajasekharan, S., Rajasekharan, R., Vaidyanathan, C.S., 1990. Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-monooxygenase from Micrococcus. Arch. Biochem. Biophys. 278, 21–25. Rani, M., Prakash, D., Sobti, R.C., Jain, R.K., 1996. Plasmid-mediated degradation of o-phthalate and salicylate by a Moraxella sp. Biochem. Biophys. Res. Commun. 220, 337–381. Robson, N.D., Parrott, S., Cooper, R.A., 1996. In vitro formation of a catabolic plasmid carrying Klebsiella pneumoniae DNA that allows growth of Escherichia coli K-12 on 3-hydroxybenzoate. Microbiology 142, 2115–2120. Rost, B., 1996. PHD: predicting one-dimensional protein structure by profile based neural networks. Methods Enzymol. 266, 525–539. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: a laboratory manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York. Sonnhammer, E.L., von Heijne, G., Krogh, A., 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology. American Association of Atificial Intelligence, Menlo Park, CA, pp. 175–182. Sua ´rez, M., Ferrer, E., Garrido-Pertierra, A., Martin, M., 1995. Purification and characterization of the 3hydroxybenzoate 6-monooxygenase from Klebsiella pneumoniae M5a1. FEMS Microbiol. Lett. 126, 283–290. Sumathi, S., Dasgupta, D., Vaidyanathan, C.S., 1998. Chemical modification of 3-HBA 6-monooxygenase by
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acterization of a gentisate 1, 2-dioxygenase from Sphingomonos sp. Strain RW5. J. Bacteriol. 180, 4171–4176. Yu, Y.M., Wang, L.H., Tu, S.C., 1987. Pseudomonas cepacia 3-hydroxybenzoate 6-monooxygenase: stereochemistry, isotope effects, and kinetic mechanism. Biochemistry 26, 105–1110. Zhou, N.Y., Fuenmayor, S.L., Williams, P.A., 2001. nag Genes of Ralstonia (formerly Pseudomonas) sp. Strain U2 encoding enzymes for gentisate catabolism. J. Bacteriol. 183, 700–708. Zhou, N.Y., Al-Dulayymi, J., Baird, M.S., Williams, P.A., 2002. Salicylate 5-hydroxylase from ralstonia sp. strain u2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase. J. Bacteriol. 184, 1547–1555.
www.elsevier.de/micres
Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 D.Q. Liua,b,c, H. Liua, X.L. Gaoa, D.J. Leakd, N.Y. Zhoua, a
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China c Graduate School of Chinese Academy of Sciences, Beijing 100039, China d Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK b
Accepted 24 September 2004
KEYWORDS 3-hydroxybenzoate 6-hydroxylase; Site-directed mutagenesis; Klebsiella pneumoniae M5a1
Summary 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis. & 2004 Elsevier GmbH. All rights reserved.
Introduction Klebsiella pneumoniae M5a1, a soil bacterium, used extensively in molecular genetic studies of dinitrogen fixation (Dixon, 1984) and various kinds of genetic manipulation, was found to be able to degrade many aromatic compounds including some hydroxybenzoates. The genes encoding the enzymes that convert 3-hydroxybenzoate (3-HBA) to pyruvate and fumarate via the gentisate pathway
were located on an 8 kb SphI fragment of K. pneumoniae M5a1 DNA (Robson et al., 1996). This fragment was sequenced in both directions, revealing seven complete open reading frames (Gao, 2003). By cloning, expression and biochemical analysis of the fragment, four of these genes (mhbMDIH) were shown to encode the enzymes involved in the catabolism of 3-HBA to fumarate and pyruvate through the gentisate pathway, of which MhbM is a 3-HBA 6-hydroxylase that catalyzes
Abbreviations: 3-HBA, 3-hydroxybenzoate; MhbM, 3-hydroxybenzoate 6-hydroxylase encoded by mhbM from Klebsiella pneumoniae M5a1; IPTG, isopropyl-b-D-thiogalactopyranoside Corresponding author. Tel./fax: 86 27 87197655. E-mail address: [email protected] (N.Y. Zhou). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.09.003
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the para-hydroxylation of 3-HBA to 2, 5-dihydroxybenzoate (gentisate), consisting of 397 amino acids with a predicted mass of 43.9 kDa (Gao, 2003). 3-HBA 6-hydroxylase (or 3-HBA 6-monooxygenase) [EC 1.14.13.24] from K. pneumoniae M5a1 is a member of the family of NAD (P) H-dependent flavoprotein hydroxylases (Sua ´rez et al., 1995). Unlike other hydroxylases which catalyze orthohydroxylation, 3-HBA 6-hydroxylase introduces a new hydroxyl group at the para-position to the existing one and yields gentisate (2, 5-dihydroxybenzoate) (Fig. 1), an intermediate step in the degradation of aromatic compounds in soil bacteria (Groseclose et al., 1973; Wang et al., 1987; Yu et al., 1987; Rajasekharan et al., 1990; Sua ´rez et al., 1995). In contrast to the ortho-hydroxylation, few studies were carried out about the para-hydroxylation during the last two decades (Zhou et al., 2001). However, gentisate and substituted gentisates are key intermediates in the aerobic pathway for the metabolism of a large number of aromatic compounds, including 3-hydroxybenzoate (Jones and Cooper, 1990; Goetz and Harmuth, 1992), substituted phenols (Crawford and Frick, 1977;
Poh and Bayly, 1980; Jain, 1996), naphthalene (Monticello et al., 1985; Grund et al., 1992; Fuenmayor et al., 1998), salicylate (Ohmoto et al., 1991; Rani et al., 1996), and 3, 6-dichloro-2methoxybenzoate (Werwath et al., 1998). Till now, 3-hydroxybenzoate 6-hydroxylase has been purified from K. pneumoniae M5a1 (Sua ´rez et al., 1995), Pseudomonas cepacia (Wang et al., 1987; Yu et al., 1987), Micrococcus sp. (Rajasekharan et al., 1990), and P. aeruginosa (Groseclose et al., 1973). Their properties were summarized in Table 1. However, no DNA sequences available so far except the mhbM from K. pneumoniae M5a1, which has also been functionally expressed. As little is known about the mechanism of parahydroxylation about this enzyme, it will be interesting to define the amino acid residues which are involved in catalytic activity of 3-HBA 6-hydroxylase. However, the three-dimension crystal structure of this enzyme is not available yet. To identify the residue which is critical for catalysis, sitedirected mutagenesis was carried out to replace Arg with Glu at position 169 in the strictly conserved motif among related enzymes that were alighted. The effects of the mutant on the activity
3-hydroxybenzoate 6-hydroxylase MhbM COOH
O2
COOH
H2O
OH
OH
NAD(P)H + H+ NAD(P) +
3-hydroxybenzoate
HO Gentisate
Figure 1. 3-hydroxybenzoate 6-hydroxylase catalyzes para-hydroxylation of 3-hydroxybenzoate to form gentisate in K. pneumoniae M5a1.
Table 1.
Comparison of 3-hydroxybenzoate 6-hydroxylases (or monooxygenases) from different strains
Enzyme
Strain
Mw(kDa)
Electron donor
Subunits
Prosthetic group
Optimal pH
3-hydroxybenzoate 6-monooxygenase 3-hydroxybenzoate 6-monooxygenase
P. cepacia
44.0
Monomer
FAD
8.0
P. aeruginosa
85.0
Monomer
FAD
8.0
3-hydroxybenzoate 6-monooxygenase
Micrococcus sp.
85.0
Monomer
FAD
8.2
3-hydroxybenzoate 6-monooxygenase
K. pneumoniae M5a1
42.0
NADH or NADPH NADH, preferred to NADPH NADH, preferred to NADPH NADH or NADPH
Monomer
FAD
8.0
ARTICLE IN PRESS Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1
of this enzyme were analyzed by color and spectrophotometric assays.
Materials and methods Strains, plasmids and general methods Escherichia coli Rosetta (DE3) pLysS (Novagen, USA) were used as the recipient strains for recombinant plasmids, which contains tRNAs for rare codons in E. coli Rosetta (DE3) pLysS. Rosetta cells were grown in LB medium at 37 1C; Kanamycin and chloramphenicol were added to final concentrations at 60 and 34 mg/ml, respectively for plasmid selection. IPTG and 3-HBA were obtained from Promega and Sigma, respectively. PyrobestTM DNA Polymerase (high fidelity), restriction and ligation enzymes were supplied by Takara Company (Dalian, China), and used as recommended by the manufacturers. DNA preparation of genes cloned into pET28a(+) (Novagen, USA), agarose gel electrophoresis, restriction enzyme digestion, DNA ligation and transformation were done by standard procedures (Sambrook et al., 1989). Purification of PCR products and DNA fragments from agarose gel was done by using the PCR purification kit from Xinzhi Biotechnology, Ltd. (Shanghai, China). Nucleotide sequences were determined by United Gene Holdings, Ltd. (Shanghai, China)
Site-directed mutagenesis The mhbM gene encoding the 3-HBA 6-hydroxylase from K. pneumoniae M5a1 was amplified from the 8 kb SphI fragment of K. pneumoniae M5a1 DNA (Robson et al., 1996) by PCR using PyrobestTM DNA Polymerase. The following oligonucleotide primers were used for this purpose: (1) 50 GCTTAGGCATTCATATGGCAAAAGTGAT30 , and (2) 50 AGGCTACGAATTCACCGCTATCTGC30 . The NdeI and EcoRI site were introduced into the primers (underlined), respectively in primers 1 and 2. The amplified PCR product was then excised with NdeI and EcoRI, ligated into the expression vector pET28a(+), and transformed into E. coli DH5a to produce plasmid pZWGM28. Overlap extension PCR was performed to introduce mutations in mhbM using the method described by Pogulis et al. (1996). Primers used to create the mutant R169E were designed as follows: (3) 50 GGTGGTGGAGCAAAGTCTGCTGG30 , and (4) 0 5 CAGACTTTGCTCCACCACCGACTTAC30 . The nucleotides mutated are underlined. The inserts of the
55
mhbM wild-type and its mutant’s clones were sequenced in both directions to ensure no mutation (for pZWGM28) or unintended mutation (for R169E) had been incorporated during the PCR prior to expression and characterization studies.
Expression of wild-type and mutant enzymes in Rosetta Rosetta cells carrying pET28a(+) containing either the wild-type or mutant insert were grown at 37 1C with shaking in LB broth with Kanamycin and chloramphenicol. When the OD600 reached 0.6, IPTG was added to final concentration of 1 mM and incubation continued for 4 h before the cells were harvested by centrifugation. Cell extracts were prepared by resuspending the bacterial pellets in ice-cold 25 mM phosphate buffer (pH 7.5) and disruption by sonication in an ice-water bath for 30 min (5 s burst per 2 s cooling), after which cell debris was removed by centrifugation at 15,000g for 30 min at 4 1C.
Enzyme assay The 3-HBA 6-hydroxylase activity assay was performed at 30 1C by measuring the rate of decrease in absorbance at 340 nm due to the substratedependent oxidation of NADH (Wang et al., 1987; Zhou et al., 2002). The sample cuvette contained 50 mM potassium phosphate buffer (pH 8.0), 0.05 mmol 3-HBA and 0.15 mmol NADH in 1-ml volume. The reference cuvette contained the same except the substrate. The assay was initiated by adding the component of 3-HBA 6-hydroxylase to both cuvettes. The specific activity of 3-HBA 6hydroxylase was calculated from the initial DA340/ min, taking the molar extinction coefficient at 340 nm for NADH as 6220 M1 cm1. Protein concentrations were determined by the Bradford method (Bradford, 1976). Specific activities are expressed as units per milligram of protein. SDSPAGE was performed according to the manufacture’s instructions (Bio-Rad). Qualitative assay was also carried out by color assay. In this assay, 3-HBA, IPTG, Kanamycin, and chloramphenicol were added to LB agar plates with final concentrations of 2 mM, 1 mM, 60 mg/ml, and 34 mg/ml, respectively. Rosetta cells carrying wildtype mhbM or its mutant clone were streaked on above plates and incubated at 30 1C overnight. An accumulation of the brown oxidation products of gentisate (Fuenmayor et al., 1998) will be observed when a clone carrying a functional mhbM gene that
ARTICLE IN PRESS 56
D.Q. Liu et al.
catalyzes the para-hydroxylation of colorless 3HBA.
Biotransformation Biotransformation (modified from Zhou et al., 2002) of 3-HBA was performed by growing Rosetta cells carrying wild-type mhbM and mutant R169E in 200 ml of LB medium containing 2 mM substrate. Kanamycin(60 mg/ml), chloramphenicol (34 mg/ml), and IPTG (1 mM) were also present in the medium. The cultures were incubated with shaking at 150 rpm and 30 1C. The cleared samples were checked periodically until the spectra of the sample had no change (between 24 and 36 h).
Computer analysis of protein sequence Nucleotide and amino acid sequence similarities were determined by BLAST-N and PSI-BLAST Searches (Altschul et al., 1990, 1997). Protein motifs were identified by BLAST-P (Altschul et al., 1997) and BLOCKs (Henikoff and Henikoff, 1994). Secondary structure prediction and analysis were performed by PHD (Rost, 1996) and TMHMM (Sonnhammer et al., 1998). Multiple sequence alignment was assembled by CLUSTAL-W (Thompson et al., 1994).
Nucleotide sequence accession number The DNA sequence of 1194 bp of the complete mhbM gene has been submitted to GenBank under accession number AY648560.
Results Constructing of mutant R169E of mhbM By amino acids sequence multiple alignments with several closely related enzymes, a high conservation of the serine and arginine was found at positions 166 and 169 of 3-HBA 6-hydroxylase from K. pneumoniae M5a1 (as shown in Fig. 2). The block K. pneumoniae M5a1 P. putida S-1s P. putida P. stutzeri Sphingomonas sp.
searcher and motif analysis showed Ser166 and Arg169 formed the highly conservative motif SXXR, which was putatively to belong to the monooxygenase family which utilizes FAD. The strictly conserved amino acids of this motif were expected to play an important role in the structure and function of the enzyme. It has been reported that an arginine residue participates the binding of 3-HBA to 3-HBA 6hydroxylase from Micrococcus sp, although it did not define which Arg was involved (Sumathi et al., 1998); A study on salicylate hydroxylase from P. putida (EC 1.14.13.1), an FAD-containing monooxygenase, also suggested that one arginine residue of the enzyme is responsible for the NADH binding site (Suzuki and Ohnishi, 1990). Therefore, sitedirected mutagenesis was carried out to obtain the mutant mhbM gene containing the aimed substitution of amino acid at position 169, from arginine to glutamate. The overlap PCR was performed using primers as described in section Materials and methods. Wild-type and mutated fragments which encoding the enzyme was inserted to T7 promoter directed plasmid pET28a(+). DNA sequencing confirmed that no mutation had been incorporated during the PCR for mhbM wild-type clone but there were indeed desired mutations created in mutant R169E (CGC to GAG).
Expressions of the wild-type and mutant enzymes and activity assays Expressions of the wild-type and mutant enzymes were performed at 37 and 30 1C. SDS-PAGE of the cell extracts (Fig. 3) showed elevated levels of a polypeptide of 44 kDa from both wild-type and mutant R169E, as expected from the deduced amino acid composition of MhbM. They are also predominantly present in the soluble form from the cell induced at 30 1C (data not shown). However, spectrophotometric assay showed that R169E mutant had completely lost enzyme activity, but the wild-type enzyme displayed 3-HBA-6-hydroxylase at a specific activity of 0.53 U/mg under the same condition.
DDVTV-FDDKGNSWTADILIGCDGGKSVVRQSLLGDSPRV------TGHV DELQVLFRD-GTEYRCDLLIGRDGIKSALRSYVLEGQGQDHLEPRFSGTC EGVTLNFAD-GSTYTADVAIAADGIKSSMRNTLLRAAGHDAVHPQFTGTS EQVVLHFKDGSKA-EADIVIGADGMRSTVRNLMLGYEDYI-----YAGYT DGVELRFAGRDPV-IADVVIGADGVRSVIRDFVTGGRAAL-----YSGTS
183 191 187 160 179
Figure 2. Multiple amino acids sequence alignment of the MhbM and its related enzymes. Aligned sequences are 3hydroxybenzoate 6-hydroxylase from K. pneumoniae M5a1; Salicylate hydroxylase from P. putida S-1 s (accession number BAA61829); Salicylate hydroxylase from P. putida (accession number Q53552); Salicylate hydroxylase from P. stutzeri (accession number AAD02157); Salicylate hydroxylase from Sphingomonas sp (accession number BAA19150). Conserved residues are shaded in black.
ARTICLE IN PRESS Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1
Figure 3. SDS-PAGE of overexpressed 3-HBA 6-hydoxylase (MhbM) and its mutant R169E in E. coli on a 12% gel. Lane 1, cell extracts containing MhbM obtained after induction with IPTG; lane 2, marker; lane 3, pET28a(+) alone; lane 4, E. coli Rosetta (DE3) pLysS alone; lane 5, cell extracts containing mutant R169E obtained after induction with IPTG. Marker masses are indicated on the left in kilodaltons. The molecular mass of the overexpressed protein (indicated by the large arrow on the right) is 44 kDa.
Qualitative color assay also showed the media surrounding the cell carrying the wild-type mhbM had changed to a brown color, indicating presence of a functional 3-HBA 6-hydroxylase; whereas no accumulation of the brown color on the plates containing the mutant, implying the mutant being no longer able to convert 3-HBA to gentisate. In the biotransformation studies, after 24 h the E. coli cells carrying wild-type 3-HBA 6-hydroxylase converted 3-HBA with a maximum wavelength (lmax) of 290 nm to a product with lmax of 320 nm, a characteristic of gentisate (Fuenmayor et al., 1998). But the mutant R169E could not transform 3HBA to the product formed from the wild-type enzyme and the spectrum of gentisate in the culture remained the same during the course.
Discussion 3-HBA 6-hydroxylase is an important enzyme in the gentisate pathway of 3-HBA degradation by catalyzing para-hydroxylation. Another example catalyzing para-hydroxylation in gentisate pathway is a salicylate 5-hydroxylase from Ralstonia sp. strain U2, which converts salicylate to gentisate. But it is
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a three-component system (a/b oxygenase components, ferredoxin reductase and ferredoxin) (Zhou et al., 2002) rather than being in the singlecomponent flavoprotein hydroxylases family, which 3-HBA 6-hydroxylase from K. pneumoniae M5a1 belongs to. Based on the presence of a highly conservative motif in MhbM and related FADcontaining monooxygenases, and evidences that an arginine is important for the activities of these enzymes for the enzymes’ activity, Arg169 was chosen for site-directed mutagenesis studies. Three independent enzyme activity assays clearly suggested that the substitution of arginine to glutamate at position 169 in the 3-HBA 6hydroxylase had resulted in the complete loss of its catalytic activity. This indicates Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis. After the Arg residue, a basic amino acid, substituted with Glu, a negative charged amino acid, both the charged character and ionized state of the residue at position 169 may lead to the failure of forming salt bond between 3-HBA and 3HBA 6-hydroxylase. Previous chemical modification investigations have suggested that an arginine residue participates the binding of 3-HBA to 3HBA 6-hydroxylase (from Micrococcus sp.) through electrostatic interaction (Sumathi et al., 1998), but it did not define which Arg was involved, apparently due to lack of sequence information. From the evidence described previously, Arg169 in MhbM may be a very important residue that participates the binding of 3-HBA to the enzyme. Another study on salicylate hydroxylase (converting salicylate to catechol) from P. putida, one of related enzymes to 3-HBA 6-hydroxylase, showed that an arginine residue at the active site of this enzyme not only acts as an NADH binding site, but also plays an essential role in maintaining a conformational state for the oxygenation of enzyme-substrate (Suzuki and Ohnishi, 1990). As to the Arg169 of 3-HBA 6hydroxylase, more experiments are needed to determine whether it is also involved in the NADH binding site. Secondary structure analysis showed no conformational transformation of MhbM was induced, after Arg was changed to Glu at position 169. The loss of enzyme activity was indicative of the importance of Arg169 in the functionality of 3-HBA 6-hydroxylase. However, when a highly conserved residue Arg 166 (equivalent to Arg169 in MhbM) was substituted with Glu in 4-hydroxybenzote hydroxylase (converting 4-hydroxybenzote to 3, 4-dihydroxybenzote) from P. fluorescens, it was found to have caused structural perturbations, and impaired binding of NADPH and FAD (Eppink et al., 1999). A further study on the structure analysis of MhbM will
ARTICLE IN PRESS 58 then be necessary to clarify the function of this or other residues essential to this important enzyme.
Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 30170036) and by the ‘‘Hundred Talents’’ program from Chinese Academy of Sciences to NYZ. We also thank Mrs. S. J. Wang for her technical assistance.
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