PEDS Advance Access originally published online on November 22, 2005
Protein Engineering Design and Selection 2006 19(1):17-25; doi:10.1093/protein/gzi071
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Enzymatic activity of Campylobacter jejuni hippurate hydrolase
1Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada NIH 8J7, 2Department of Food Science and 3Pathobiology Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and Departments of 4Medical Genetics and Microbiology and 5Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
6 To whom correspondence should be addressed. E-mail: jodumeru{at}lsd.uoguelph.ca
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Abstract |
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The hippurate hydrolase enzyme of Campylobacter jejuni was expressed in Escherichia coli as a six-histidine-tagged fusion protein. The purified recombinant enzyme was characterized to gain an understanding of the structure and activity of the hippurate hydrolase. The recombinant enzyme had a native molecular mass of 193 ± 11 kDa a reduced molecular mass of 42.4 ± 0.8 kDa, and possessed 1.98 ± 0.68 molecules of zinc per enzyme subunit molecule, suggesting that it was a homotetramer with two associated zinc ions. The enzyme was a metallocarboxypeptidase that was sensitive to silver, copper and ferrous ions, and displayed optimal activity at pH 7.5 and 50°C. It hydrolyzed carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic amino acids. A high proportion of the enzyme structure consisted of highly ordered
-helix and ß-sheet sequences. An alignment of the amino acid sequence of the hippurate hydrolase enzyme with those of related enzymes with similar activities revealed several conserved amino acids, which might be involved in enzyme catalysis or metal ion binding for the enzyme. Site-directed mutagenesis of the recombinant enzyme demonstrated that the Asp76, Aps104, Glu134, Glu135, His161 and His356 positions were important for the catalytic activity of the enzyme.
Keywords: amidohydrolase/carboxypeptidase/hippurate hydrolase/mutagenesis
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Introduction |
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Campylobacter species are predominant causes of bacterial gastroenteritis in North America (Altekruse et al., 1999
; CDC, 2002). Campylobacter jejuni is responsible for 
90% of Campylobacter isolated from humans with gastroenteritis, while other species, such as Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter hyointestinalis, account for only a small proportion of the isolates (Tauxe, 1992). The ability of C.jejuni to hydrolyze hippurate (N-benzoylglycine) to benzoic acid and glycine is commonly used to distinguish C.jejuni from other Campylobacter species, which lack this characteristic (Nachamkin, 1995). Hippurate hydrolase activity in C.jejuni is due to the presence of a species-specific hippurate hydrolase enzyme, encoded by the hipO gene (Hani and Chan, 1995).
The hippurate hydrolase enzyme of C.jejuni was classified as a non-peptidase homologue of the M40 peptidase family within the MH peptidase clan by the Merops Protease Database, on the basis of amino acid homology between the hippurate hydrolase and other members of this family (Merops, 2002). Other M40 family peptidases are found in plants, archaebacteria and eubacteria (Merops, 2002). The hippurate hydrolase of C.jejuni may be considered an amidohydrolase on the basis of its ability to cleave the benzoyl group from N-benzoylglycine. Other M40 peptidases were demonstrated experimentally to be amidohydrolases, including a thermostable carboxypeptidase from Sulfolobus solfataricus (Colombo et al., 1992, 1995), an N-acyl-L-amino acid amidohydrolase from Bacillus stearothermophilus (Sakanyan et al., 1993), a family of indole-3-acetic acid (IAA) amino acid hydrolases from the plant Arabidopsis thaliana (Bartel and Fink, 1995; Davies et al., 1999), an IAA-aspartic acid hydrolase from Enterobacter agglomerans (Chou et al., 1998) and an N-carbamylase from Pseudomonas sp. (Watabe et al., 1992). The amino acid sequence identity of these M40 peptidases with the C.jejuni hippurate hydrolase ranged from 36% identity for the S.solfataricus and B.stearothermophilus enzymes to 25% identity for the IAA-aspartic acid hydrolase from E.agglomerans.
No tertiary structures have been elucidated for the M40 peptidases, but amino acids were identified in the Merops Protease Database that were proposed to contribute to zinc-binding or catalysis for M40 peptidases, on the basis of amino acid sequence conservation among these enzymes (Merops, 2002). A separate study identified amino acids in the thermostable carboxypeptidase of S.solfataricus that were proposed to contribute to zinc-binding and enzyme catalysis, on the basis of sequence conservation between this enzyme and a large group of similar enzymes termed the zinc peptidase superfamily (Makarova and Grishin, 1999). This superfamily included not only peptidases but also N-acetylases and N-desuccinylases. An investigation of the contributions of the residues identified to enzyme activity may yield insights into the active sites of these enzymes. Tertiary structures for peptidases of the related M20 and M28 families, within the MH clan, were elucidated by X-ray crystallography (Chevrier et al., 1994, 1996; Greenblatt et al., 1997; Rowsell et al., 1997; Hakansson and Miller, 2002). Other enzymes may also have conserved tertiary structures (Jozic et al., 2002; Lundgren et al., 2003).
The in vivo function of the hippurate hydrolase of C.jejuni remains undetermined. Hippurate hydrolase activities were reported in several other microorganisms, and hippurate hydrolase enzymes from Pseudomonas spp., Fusarium semiticum and Rhodococcus equi have been purified and characterized (Kameda et al., 1968; Rohr, 1968a,b,c; Ottow, 1974; Miyagawa et al., 1985, 1986). Unfortunately, the functions of these purified enzymes were not determined, and their amino acid sequences are not available for comparison with that of the C.jejuni hippurate hydrolase. It is not known whether hippurate hydrolases from different microorganisms perform similar functions or not. The clusters of orthologous domains (Tatusov et al., 1997, 2001; COG, 2002) database (COG, 2002), which categorizes proteins from sequenced microbial genomes into phylogenetic lineage groups, placed the C.jejuni hippurate hydrolase in group R, which contains proteins of poorly characterized function.
The activity of the hippurate hydrolase enzyme of C.jejuni, both within its host bacterium and as a recombinant enzyme, can be easily measured (Hani and Chan, 1995; Nachamkin, 1995), making the enzyme an ideal subject for study of the mechanisms of M40 peptidases in general. This information should also yield insight into the role that the hippurate hydrolase enzyme plays within C.jejuni and whether this enzyme contributes to the higher frequency at which C.jejuni is isolated from cases of human enteritis, compared with other Campylobacter species. The first objective of this study was to characterize the physical and enzymatic properties of the C.jejuni hippurate hydrolase, to gain information on the types of reactions this enzyme might catalyze and to compare these properties with those of other M40 peptidases and hippurate hydrolases from other species. The second objective was to evaluate the contribution of conserved amino acids to the zinc-binding and enzymatic activity of the C.jejuni hippurate hydrolase and to gain information about the active sites of the hippurate hydrolase and other M40 peptidases. Identification of active site residues would be useful for elucidating tertiary structures of the C.jejuni hippurate hydrolase and other M40 peptidases.
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Materials and methods |
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Plasmid vectors and bacterial stains
C.jejuni ATCC 43 431 was obtained from Dr J. L. Penner, formerly of the University of Toronto. The plasmid pHipO was prepared previously (Hani and Chan, 1995). The plasmid pQE30 and Escherichia coli M15[pREP4] were obtained from Qiagen (Valencia, CA).
Preparation of recombinant hippurate hydrolase
The hipO gene was amplified by PCR using primers (5'-CTCGGATCCATGAATTTAATTCCAGAA-3' and 5'-GAGGTCGACTTATTTTAAGTATTTTAAAG-3') based on its published gene sequence (Hani and Chan, 1995), while introducing BamHI and SalI sites on either side of the gene, and using the pHipO plasmid for template DNA. The hipO gene was subcloned into the BamHI and SalI sites of the pQE30 vector to create a type IV construct and the construct was expressed in E.coli M15[pREP4], as described previously (Qiagen, 1996). The fusion protein was purified under non-denaturing conditions by nickel-NTA affinity chromatography, as described previously (Qiagen, 1996).
Microcon YM-10 centrifugal filter devices (Millipore Corporation; Bedford, MA) were used to exchange the elution buffer with 10 mM phosphate buffer, pH 8.0, for enzyme preparations used in enzyme activity and circular dichroism studies. Enzyme preparations used to determine metal ion concentrations were desalted by dialysis at room temperature against three changes of deionized water. Enzyme preparations used for metal ion analysis and circular dichroism studies were prepared fresh before the analysis, while enzyme preparations used to study enzyme activity were stored at –20°C in 50% glycerol. Enzyme blank solutions were prepared in the same manner using E.coli M15[pREP4]. Protein concentrations were determined by the BCA protein assay (Pierce Biochemicals, Rockford, IL).
Size, subunit arrangement and metal composition of recombinant hippurate hydrolase
The reduced molecular mass of the recombinant enzyme was determined on Coomassie blue R250-stained SDS–PAGE gels. The native molecular mass of the enzyme was calculated by gel permeation chromatography performed at RT on Sephadex G-200 resin (Amersham Biosciences Corp., Baie d'Urfe, QC), using 50 mM sodium phosphate, pH 7.0, 100 mM NaCl and 0.01% NaN3 as the mobile phase. Broad-range ion content (Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Si, Sr, Zn) of the purified enzyme and blank solutions was determined by inductively coupled plasma emission (ICP) analysis using a Thermo Jerrel Ash IRIS ICP atomic emission spectrometer. The molar ratios of zinc to the enzyme subunit in duplicate enzyme preparations were calculated by assuming a theoretical subunit molecular weight of 43 558 Da for hippurate hydrolase [determined using the Compute pI/Mw program, which calculates molecular mass of proteins by the average isotopic masses of their constituent amino acids (ExPASy, 2002)] and assuming 100% enzyme purity. The purity of the enzyme solutions, as measured by gel densitometry, was 99%. Reduced and native molecular mass determinations were performed twice and metal analysis was performed three times.
Circular dichroism analysis of recombinant hippurate hydrolase
Enzyme preparations were diluted to 100 µg/ml protein in 10 mM phosphate buffer and filtered through a 0.22 µm syringe filter. Far UV circular dichroism analysis was performed, under constant nitrogen purge at 23°C, using a Jasco J-600 spectropolarimeter at wavelengths of 250 to 190 nm. Each of the six recombinant enzymes and the wild-type enzyme was analyzed twice, using two separate enzyme preparations, and in each analysis the average of three readings was taken. Results of each analysis were adjusted by subtracting the enzyme blank readings for each wavelength. The proportions of -helix, ß-sheet, ß-turn and random coil in the protein samples were estimated using the Jasco J-600 DP-J-600/PS2 system version 1.41 software, copyright Jasco, 1989.
Enzyme activity of recombinant hippurate hydrolase
The hydrolytic activities of recombinant hippurate hydrolase towards different synthetic substrates were determined in 100 µl reaction volumes containing 50 mM sodium phosphate, pH 7.5, and 10 mM of each substrate. The reactions were initiated by adding 1 µl (36–180 ng) of purified recombinant enzyme diluted in 0.85% w/v NaCl, and allowed to proceed for 10 min at 37°C. Hydrolysis of p-nitroanilide aminopeptidase and endopeptidase substrates by the recombinant enzyme was assayed by measuring the absorbencies of these solutions at 410 nm using a Beckman DU 650 spectrophotometer. Hydrolysis of carboxypeptidase substrates by the recombinant enzyme was determined by adding 750 µl of ninhydrin reagent (Doi et al., 1981) and incubating the mixture at 84°C for 5 min to generate a colored product (Colombo et al., 1992). Reaction mixtures were centrifuged for 1 min at 12 000 g in a microfuge before measuring color development at 505 nm, using a Beckman DU 650 spectrophotometer. All substrates were obtained from Sigma–Aldrich Canada Ltd. (Oakville, ON) except N-benzoyl substituted methionine, alanine, valine, phenylalanine, histidine, lysine, arginine and tryptophan and N-acetyl-glycine, which were obtained from Bachem BioScience Inc. (King of Prussia, PA).
The activity of the recombinant hippurate hydrolase was compared with that of the native C.jejuni enzyme, using cleared whole bacterial lysates of this bacterium as a source of the enzyme. The activity of the native enzyme was evaluated by adding 50 µl of cleared whole bacterial lysate to 50 µl of 20 mM N-benzoylglycine and 100 mM sodium phosphate (pH 7.5) and incubating the mixture for 10 min at 37°C. Enzyme activity, as a function of free glycine released, was measured as described above for carboxypeptidase substrates (Doi et al., 1981; Colombo et al., 1992). Sample blanks consisted of the sample reaction mixtures without the N-benzoylglycine substrate.
The in vitro pH optimum of the recombinant hippurate hydrolase against N-benzoylglycine was determined by replacing the 50 mM phosphate in the standard assay reaction mixture with the same molarity of sodium citrate, sodium phosphate, Tris or sodium carbonate, resulting in solutions of pH values from 2.0 to 10.8. The in vitro temperature optimum of the recombinant hippurate hydrolase against N-benzoylglycine was determined by incubating the standard assay reaction mixture at temperatures ranging from 0°C to 80°C.
The effects of peptidase inhibitors on the activity of the recombinant hippurate hydrolase against N-benzoylglycine were determined by preincubating the recombinant enzyme in 90% of the reaction volume (90 µl) in the standard assay mixture plus 2 mg ml–1 of BSA and 0.5 mM PMSF, 0.5 mM 1,10-phenanthroline, 0.5 µg ml–1 pepstatin A, 0.05 mM E64, 10 mM EDTA or 1 mM 2-mercaptoethanol (final concentrations based on a volume of 100 µl) for 30 min at 37°C. The reaction was initiated by adding 10 µl of 100 mM N-benzoylglycine. Control samples, used to estimate the activity of the enzyme without the inhibitor, contained 1% ethanol for PMSF, 1,10-phenanthroline and pepstatin A samples to account for the ethanol present from the inhibitor stock solutions.
The effect of metal ions on the activity of recombinant hippurate hydrolase against N-benzoylglycine was determined by adding 2 mM AgNO3, CuSO4, BaCl2, MgCl2, NiSO4, CoCl2, FeSO4, MnSO4, ZnCl2, CaCl2, CdCl2 or KNO3 to the standard assay mixture.
Specific activities (µmol amino acid liberated min–1 mg protein–1) of the recombinant enzyme for each substrate were determined using standard curves for each free amino acid, performed on the same day using the same batch of ninhydrin reagent. The Km and Vmax values of the activities of the enzyme towards synthetic substrates and those of whole bacterial lysates of C.jejuni ATCC 43 431 towards N-benzoylglycine were calculated using the GraFit software, version 3.01 (Microsoft Corp.). The Kcat and Kcat/Km values were determined as described previously (Copeland, 1996). Results for all enzyme reactions are the average of two experiments, each performed in duplicate (n = 4).
Comparison of amino acid sequence of hippurate hydrolase with amino acid sequences of homologous enzymes
A basic local alignment search tool for proteins (BLASTP) (Altschul et al., 1990; NCBI, 2002) was used to search for proteins homologous to the C.jejuni hippurate hydrolase, using the translated amino acid sequence of hipO as a query sequence. The search identified several homologous amidohydrolases or putative amidohydrolases, including the thermostable carboxypeptidase from S.solfataricus, the thermostable aminoacylase from B.stearothermophilus and two IAA-amino acid hydrolases from A.thaliana, which were M40 peptidases shown to have amidohydrolase activity. Amino acid sequences of these M40 peptidases were compared with that of the hippurate hydrolase enzyme, using the ClustalW program (Thompson et al., 1994). Amino acids from C.jejuni hippurate hydrolase that aligned with amino acids proposed to play a role in enzyme catalysis and zinc-binding of the thermostable carboxypeptidase from S.solfataricus (Makarova and Grishin, 1999; Merops, 2002) were identified.
Preparation of site-directed mutants of recombinant hippurate hydrolase enzyme
Site-directed mutagenesis of the hipO gene was performed using the QuikChange® Site-directed mutagenesis kit (Stratagene; La Jolla, CA), using the pQE30-hipO construct, prepared above, as a template. The amino acids that were mutated and the primers used to create the mutants are as follows: Asp76
Asn76 (F: 5'-GGACTTCGTGCAAATATGGATGCTTTGCCTTTGC-3' and R: 5'-GCAAAGGCAAAGCATCCATATTTGCACGAAGTCC-3'), Asp104Asn104 (F: 5'-GTAATGCATGCTTGCGGTCATAATGGACATACTACTTC-3' and R: 5'-GAAGTAGTATGTCCATTATGACCGCAAGCATGCATTAC-3'), Glu134Gln134 (F: 5'-CTTTATTTTCAACCTGCTCAAGAGGGTTTGGGTGGTGC-3' and R: 5'-GCACCACCCAAACCCTCTTGAGCAGGTTGAAAATAAG-3'), Glu135Gln135 (F: 5'-CTTTATTTTCAACCTGCTGAACAGGGTTTGGGTGGTGC-3' and R: 5'-GCACCACCCAAACCCTGTTCAGCAGGTTGAAAATAAAG-3'), His161Ala161 (F: 5'-GTGATTATGTTTTTGGATGGGCCAATATGCCTTTTGGTAGCG-3' and 5'-CGCTACCAAAAGGCATATTGGCCCATCCAAAAACATAATCAC-3') andHis356Ala356 (F: 5'-GTGATTATGTTTTTGGATGGGCCAATATGCCTTTTGGTAGCG-3' and R: 5'-CGCTACCAAAAGGCATATTGGCCCATCCAAAAACATAATCAC-3'). The mutant enzyme constructs were sequenced to confirm that the desired mutations were present. Purified mutant recombinant enzymes were prepared as described for the wild-type recombinant hippurate hydrolase. An enzyme blank solution was prepared with each batch of enzyme. Circular dichroism analysis and enzyme activity towards N-benzoylglycine of the mutant recombinant hippurate hydrolases were determined as described for the wild-type recombinant enzyme.
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Structure and metal-binding of the recombinant hippurate hydrolase enzyme
The purified recombinant hippurate hydrolase had a reduced molecular mass of 42.4 ± 0.8 kDa on Coomassie blue-stained SDS–PAGE gels and a native molecular mass of 193 ± 11 kDa, calculated by size exclusion chromatography, suggesting that the hippurate hydrolase was a homotetramer of identical 42.4 kDa subunits. Detectable levels of sulfur and zinc ions were found, by ICP analysis, to be present in recombinant hippurate hydrolase solutions but not in enzyme blank solutions. The sulfur was attributed to sulfur-containing amino acids in the recombinant protein while the zinc was assumed to represent a metal ion cofactor. Enzyme solutions contained a molar ratio of 1.98 ± 0.68 molecules of zinc per enzyme subunit. Secondary structure predictions, based on the far UV circular dichroism analysis of the recombinant hippurate hydrolase, suggested that the enzyme possessed 26.3 ± 2.9% of -helix, 40.0 ± 5.1% ß-sheet and 11.4 ± 1.3% ß-turn secondary structures and 22.2 ± 1.0% random coil.
Enzymatic activity of the recombinant hippurate hydrolase enzyme
The substrate specificity of the purified recombinant enzyme was tested using a variety of commercially available synthetic substrates. The results are summarized in Table I. The enzyme showed the highest specificity towards N-benzoylamino acids with the small aliphatic amino acids glycine, alanine, and valine or the sulfur-containing amino acid methionine in the P1' position and did not hydrolyze typical substrates for aminopeptidases (lysine-p-nitroanilide, leucine-p-nitroanilide) or endopeptidases (N-benzoylarginine-p-nitroanilide, succinyl-phenylalanine-p-nitroanilide). The Km values for the enzyme activities of the purified recombinant hippurate hydrolase and the native hippurate hydrolase, tested using whole bacterial lysates of C.jejuni ATCC 43 431, towards N-benzoylglycine were 764 ± 57 and 650 ± 42, respectively, suggesting that the six-histidine tag did not affect the affinity of the enzyme towards this substrate. N-benzoylglycine was used as a substrate in all subsequent assays.
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The pH and temperature optima of the purified recombinant hippurate hydrolase against N-benzoylglycine were found to be 7.5 and 50°C, respectively. The effects of peptidase inhibitors and metal ions on the activity of the purified recombinant hippurate hydrolase are summarized in Figure 1. The enzyme activity of the recombinant hippurate hydrolase was strongly inhibited by 0.5 mM of the metallopeptidase inhibitor 1,10-phenanthroline (0.8 ± 1.0% of control activity), but was not significantly inhibited by 0.5 mM PMSF, 0.5 µg ml–1 pepstatin A, 0.05 mM E64 or 1 mM 2-mercaptoethanol, suggesting that the enzyme was a metallopeptidase. Interestingly, the enzyme activity was slightly enhanced in the presence of 10 mM EDTA (123.9 ± 27.4%). The enzyme was strongly inhibited by silver (1.7 ± 0.3%) and copper (21.9 ± 16.1%) ions and moderately inhibited by ferrous, nickel, cobalt, manganese, zinc, calcium and cadmium ions (32.7–53.4%), but barium, magnesium and nitrate ions were not inhibitory (113.7–123.3%).
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Identification of amino acids important for enzyme activity and/or zinc-binding of the recombinant hippurate hydrolase enzyme
A ClustalW alignment of the amino acid sequences of the hippurate hydrolase enzyme of C.jejuni, the thermostable carboxypeptidase from S.solfataricus, the thermostable aminoacylase from B.stearothermophilus and two IAA-amino acid hydrolases from A.thaliana is shown in Figure 2. Several amino acids were conserved between the enzyme sequences, including two regions of seven amino acids each (RADMDAL and MHACGHD). The Asp81, Asp109, Glu142 and His365 amino acids of the thermostable carboxypeptidase from S.solfataricus were proposed, in the Merops Protease Database, to bind zinc ions (Merops, 2002). These aligned with the Asp76, Asp104, Glu135 and His356 amino acids of the hipO gene product. The Asp83 and Glu141 amino acids of S.solfataricus were predicted by the Merops Protease Database to be located in the enzyme active site. These residues aligned with the Asp78 and Glu134 residues of the hipO gene product. The Asp81, Asp109, Glu141, His168 and His365 amino acids of the thermostable carboxypeptidase from S.solfataricus were proposed by Makarova and Grishin (1999) to bind zinc ions. These aligned with the Asp76, Asp104, Glu134, His161 and His356 amino acids of the hipO gene product. The Glu142 amino acid of S.solfataricus was predicted by Makarova and Grishin (1999) to be involved in enzyme catalysis. This residue aligned with the Glu135 residue of the hipO gene product.
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Site-directed mutagenesis of the amino acid residues of C.jejuni hippurate hydrolase that corresponded with those predicted to contribute to the function of the S.solfataricus enzyme by Makarova and Grishin (1999)
, Asp76, Asp104, Glu134, Glu135, His161 and His356, resulted in a complete loss of detectable Bz-Gly hydrolytic activity for Asp104, Glu134, Glu135, His161 and His356 mutants, and the Asp76 mutant displayed only 0.72 ± 0.15% of the wild-type Kcat/Km activity (Table II). Analysis of the levels of zinc associated with the mutant and wild-type enzymes (Table II) revealed that the Asp104, Glu135, His161, Val250 and His356 mutants bound significantly lower amounts of zinc than the wild-type enzyme (P < 0.05) but the Glu134 mutant was not significantly reduced in the level of zinc associated with the enzyme (P = 0.2490). The Asp76 mutant also bound lower amounts of zinc but this reduction was not statistically significant (P = 0.0997).
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The mutant hippurate hydrolase enzymes were unchanged in their migration on Coomassie blue R250-stained SDS–PAGE gels (data not shown) and their binding to monoclonal antibodies generated against the hippurate hydrolase enzyme (Steele et al., 2002
), compared with the wild-type enzyme. Circular dichroism profiles of the mutant enzymes were also very similar to those of the wild-type enzyme (Figure 3). When the proportions of -helix, ß-sheet and ß-turn secondary structures present in the enzymes were estimated, based on their circular dichroism profiles, the only statistically significant difference between the wild-type enzyme and the mutant enzymes was a reduction in the proportion of -helix structure present in the Asp76 mutant (P = 0.0444).
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The properties of the recombinant hippurate hydrolase were compared with those of hippurate hydrolases purified from Pseudomonas spp., F.semiticum and R.equi and to those of enzymes that displayed high levels of amino acid homology to the hippurate hydrolase of C.jejuni. The hippurate hydrolase enzyme was a homotetramer of 42.4 kDa subunits. Previously purified hippurate hydrolases from Pseudomonas putida and Pseudomonas sp. and the homologous carboxypeptidase from S.solfataricus had subunit sizes of 40–48 kDa, and existed as homomultimers of four or six subunits, suggesting that the subunit size and organization were conserved amongst these enzymes (Miyagawa et al., 1985
, 1986; Colombo et al., 1992; Sakanyan et al., 1993).
Secondary structure predictions, from the far UV circular dichroism profile of the enzyme, indicated that a high proportion of the enzyme occurred within secondary structures, suggesting that the enzyme had a highly ordered structure. The high temperature optimum and neutral pH optimum observed for recombinant hippurate hydrolase were also similar to those of purified hippurate hydrolases from Pseudomonas spp. F.semiticum and R.equi but slightly lower than those of homologous enzymes from S.solfataricus and B.stearothermophilus (Kameda et al., 1968; Rohr et al., 1968a,b,c; Miyagawa et al., 1985, 1986; Colombo et al., 1992). Metallopeptidases frequently display optimal activity at neutral pH values (Rao et al.,1998). While the temperature optimum of C.jejuni hippurate hydrolase was fairly high at 50°C, the temperature optima of the homologous enzymes were significantly higher. Further comparison of these enzymes might yield information on the structural characteristics that contribute to enzyme thermostability.
Inhibition of the hippurate hydrolase by the metal ion chelator 1,10-phenanthroline, but not by other peptidase inhibitors, indicated that hippurate hydrolase enzyme was a metallopeptidase. Metallopeptidases require metal ions at their active sites for activity. Metal analysis of enzyme solutions indicated that zinc was the metal ion cofactor for the enzyme. The hippurate hydrolases from P.putida and R.equi and the homologous enzymes from S.solfataricus and B.stearothermophilus are also metallopeptidases (Miyagawa et al., 1985, 1986; Colombo et al., 1992; Sakanyan et al., 1993).
The use of the metal ion chelator 1,10-phenanthroline is preferable to that of EDTA, as the affinity of 1,10-phenanthroline for zinc ions is much stronger than its affinity for alkaline earth metal ions, such as calcium (Auld, 1995). Chelation of the calcium ions might alter the tertiary structure of enzymes, leading to a non-specific loss of activity in peptidases. It is interesting that the recombinant hippurate hydrolase enzyme was not significantly inhibited by the metal ion chelator, EDTA, a phenomenon that was also observed for other purified hippurate hydrolase enzymes (Kameda et al., 1968; Miyagawa et al., 1985, 1986).
The inhibitory effects of zinc and other metal ions on enzyme activity were similar to those observed for other purified hippurate hydrolase enzymes (Kameda et al., 1968; Miyagawa et al., 1985, 1986). The slight inhibitory effect of millimolar levels of zinc ions on enzyme activity of zinc metallopeptidases has been reported previously (Larsen and Auld, 1989).
The substrate specificity displayed by the hippurate hydrolase indicated that it is a carboxypeptidase with a preference for N-acylated amino acids, with glycine, alanine, valine, methionine or glutamate in the P1' position. The enzyme did not hydrolyze substrates with the basic amino acids histidine, lysine and arginine or the aromatic amino acids phenylalanine and tryptophan in the P1' position, suggesting that the size, negative charge and/or hydrophobicity of these residues were incompatible with the hippurate hydrolase enzyme binding site.
The moiety in the P1 position of the substrate was also important as N-carbobenzyloxy-linked glycine was not cleaved at a detectable rate by the enzyme while N-benzoylglycine was rapidly hydrolyzed, and N-acetyl-linked glycine and alanine were hydrolyzed at lower rates than N-benzoyl-linked derivatives of these amino acids. The hippurate hydrolase enzymes from P.putida, F.semiticum and R.equi displayed similar substrate utilization profiles (Kameda et al., 1968; Rohr, 1968a,b, c; Miyagawa et al., 1985, 1986), while homologous enzymes from B.stearothermophilus and S.solfataricus had very different substrate specificities, favoring basic and/or aromatic amino acids in the P1' position (Colombo et al., 1992; Sakanyan et al., 1993).
The loss of N-benzoylglycine hydrolytic ability observed in the mutated hippurate hydrolase enzymes suggested that the mutated amino acid residues were important for enzyme function. The fact that the far UV circular dichroism profiles of the mutant enzymes were not significantly different from that of the wild-type enzyme suggested that these losses of activity were not due to distortions of the enzyme conformation. Tertiary structures have not been determined from members of the M40 peptidase family but structures have been published for the Vibrio aminoacylase and Streptomyces aminopeptidase enzymes of family M28 and the Pseudomonas glutamate carboxypeptidase of family M20 from within the MH clan (Barrett et al., 1998).
Zinc ligands in these structures were bound by histidine, aspartate and glutamate residues (Barrett et al., 1998). In enzymes with cocatalytic zinc ions, an aspartate or a glutamate residue frequently forms a bridge between the two metal ions (Vallee and Auld, 1993). For the Vibrio proteolytica aminopetidase of family M28, which contains cocatalytic zinc ions, Zn1 is coordinated with carboxylate oxygens of Glu152, the N
2 of His256, a bridging water molecule and a bridging carboxylate O
1 of Asp117 (Chevrier et al., 1994; Rawlings and Barrett, 2004). The Zn2 ion is bound to both carboxylate oxygens of Asp179, the N2 of His97, the bridging water molecule and the bridging carboxylate O1 of Asp117. Loss of metal-binding and catalytic activity in the mutant enzymes suggests that the Asp104 and Glu135 residues of hippurate hydrolase might be equivalent to the bridging Asp117 residue and the Zn1-binding Glu152 of the V.proteolytica aminopetidase. The Zn1-binding His256 of the V.proteolytica aminopetidase might be equivalent to either the His161 or His336 residues of the hippurate hydrolase. These residues are all located within conserved regions of the amino acid sequence (Figure 2). Other conserved amino acids may form secondary interactions that contribute to enzyme structure. These residues may be identified by further mutation studies or by X-ray crystallography.
The results of this study suggest that the hippurate hydrolase of C.jejuni is also a metallopeptidase, possibly with cocatalytic zinc ions bound by aspartate, glutamate and histidine residues. This information may be useful in elucidating the tertiary structure of the hippurate hydrolase or other peptidases of the M40 family.
In conclusion, conservation of substrate specificity and temperature optima within the hippurate hydrolase enzymes suggests that these enzymes, which were selected for by their affinity for a common substrate, share similar physiochemical characteristics. Differences in substrate specificity between the C.jejuni hippurate hydrolase and homologous enzymes from S.solfataricus and B.stearothermophilus suggest that while these enzymes may reflect a common enzyme mechanism and tertiary structure, they are unlikely to serve the same function in vivo. The metallopeptidase activity of the C.jejuni hippurate hydrolase and the presence of two associated zinc ions suggest that the hippurate hydrolase of C.jejuni was correctly placed within the M40 peptidase family, and site-directed mutagenesis studies suggested that predictions made by Makarova and Grishin (1999) with respect to conserved amino acids being important for enzyme function were accurate.
The enzymatic properties of the hippurate hydrolase enzyme suggest that it would be active under normal physiological conditions. The carboxypeptidase activity of this enzyme might contribute to nutrient acquisition by the bacterium during infection or to processing of a specific peptide component of the bacterium that contributes directly to the ability of C.jejuni to cause disease in humans. Further studies, such as the development of isogenic mutants, should establish the role of the hippurate hydrolase enzyme in C.jejuni.
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The authors would like to thank the Ontario Ministry of Agriculture and Food, Vita-Tech Inc. and the Ontario Graduate Scholarship in Science and Technology for their financial support of this project. The authors would also like to thank the staff at the Solutions Analytical Lab, University of Waterloo, Waterloo, Ontario, who performed the inductively coupled plasma emission atomic adsorption spectroscopy analysis, and the Guelph Molecular Supercenter at the University of Guelph, where the mutant enzyme constructs were sequenced.
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Received May 11, 2005; revised August 10, 2005; accepted September 12, 2005.
Edited by Alan Berry
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