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Protein & Peptide Letters, 2009, 16, 36-45
Structural Bioinformatics of Vibrio cholerae Aminopeptidase A (PepA) Monomer Ghosia Lutfullah1, Noreen Azhar1,*, Farhat Amin1, Zahid Khan1, M. Kamran Azim2, Khalida Shouqat2, Sajid Noor2 and Rizwan Ali2 1
Center of Biotechnology, University of Peshawar, Peshawar, Pakistan; 2Husein Ebrahim Jamal Research Institute of Chemistry, International Center of Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan Abstract: Aminopeptidase A (PepA) is a metalloexopeptidase found in Vibrio cholerae .It functions as a transcriptional repressor in regulatory cascade that controls virulence gene expression in V. cholerae. It is involved in protein degradation and in the metabolism of biologically active peptides. We proposed a 3D model of PepA based upon the crystal structure of PepA from Escherichia coli (E. coli) with an intention to evaluate the active site of the enzyme and to predict the properties of this enzyme, study of its 3D structure will help in understanding its role in DNA binding.
Keywords: Aminopeptidase (PepA) Vibrio cholerae, protein degradation, homohexamer, homology modeling, 3D structure, DNA binding. 1. INTRODUCTION Aminopeptidases constitute a diverse set of peptidases with important roles in cell maintenance, growth, development, and defense. Historically, aminopeptidases were regarded as a ubiquitous set of enzymes with mundane roles in cell homeostasis [1]. They are of critical biological and medical importance because of their key role in protein degradation and metabolism of biologically active peptides [2], converting the peptides released by endoproteases or the proteasome to their amino acid constituents [1]. Many but not all of these proteases are zinc metalloenzymes having bastatin and amastatin as potent inhibitors [3, 4]. They contain NTDAEGRL motif as a signature pattern for these enzymes, a conserved octapeptide which contain 2 residues involved in binding metal ions, an Asp and a Glu [5-7]. A metal-bridging water molecule is thought to act as a nucleophile in the reaction mechanism [34]. In bacteria and animals, aminopeptidases not only serve to proteolytically process the N-terminus of proteins and bioactive peptides, but many also have secondary functions, with distinctive moonlighting functions ranging from transcriptional repressors, site-specific recombination factors, viral or toxin receptors to vesicular trafficking and interacting with key membrane transporters. LAPs are often viewed as cell maintenance enzymes with critical roles in turnover of peptides. [1]. Leucine aminopeptidases (LAPs) constitute a diverse set of exopeptidases that catalyze the hydrolysis of leucine residues from the amino-termini of protein or peptide substrates [8]; notably, LAPs have distinct substrate specificities that extend beyond Leu hydrolysis. LAPs function in a range of subcellular locations, which determines substrate availability. These enzymes have variable temperature and pH optima and divalent cation requirements [1]. PepA aminopeptidases
are distinguished by their large size and hexameric structure [34]. M17 LAPs are hexameric and bind two cations.While hydrolyzing Leu substrates, LAPs often have a broader specificity. In microbes, the M17 LAPs have a role in proteolysis and have also acquired the ability to bind DNA. This property enables LAPs to serve as transcriptional repressors to control pyrimidine, alginate and cholera toxin biosynthesis, as well as mediate sitespecific recombination events in plasmids and phages [1]. Vibrio cholerae as a specie includes both pathogenic and non pathogenic strains [9], producing a variety of proteins, which perform different roles i.e., amino acid synthesis and degradation, transport energy metabolism, and DNA metabolism etc, along with its vital pathogenic activity. Aminopeptidase A (PepA) is one of the major aminopeptidase in V. cholerae and is thought to be important for the metabolism of peptides supplied exogenously or produced by protein degradation with in the cell [10]. The biochemical properties and primary sequence of PepA shows that it belongs to MF clan [8] and leucine aminopeptidase (LAP) family [11], M17 of metallopeptidases [8]. Analysis of approximately 6kb fragment in the vicinity of PepA gene revealed four open reading frames (ORFs). These ORFs encode a protein of 503 amino acids with a calculated Mol.wt of 54,617 Da and pI of 6.37 [12]. V. cholerae PepA shows peptidase activity against Leu-p-nitroanilide [13], it is the only enzyme that survives the heat treatment at 70o C in crude extracts of E. coli [34]. Disruption of PepA gene in V. cholerae results in elevated levels of expression of virulence genes at non-inducing pH [12]. PepA proteins from V. cholerae which are very similar to E. coli PepA can function in Xer recombination [34]. The present study relates to the prediction of 3D model of PepA from V. cholerae based on comparative homology modeling, enzyme mechanism and identification of important residues that contribute to active site of enzyme.
*Address correspondence to this author at the Center of Biotechnology, University of Peshawar, Peshawar, Pakistan; Tel: +92-321-9108043; + 92300-5979401; E-mail:
[email protected] 0929-8665/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.
Structural Bioinformatics of Vibrio cholerae
2. METHODOLOGY 2.1. Sequence Alignment Peptidase family M17 of V. cholerae was selected and studied [8]. Amino acid sequence of PepA was obtained from Swiss-Prot database “(AC#: P0C6E1)” [14-16]. Sequence homology searches were carried out using BLAST algorithm [17] against PDB [18]. Target template alignment was created using ALIGN2D command of Modeller [19]. 2.2. Sequence Comparison Sequences homologous to PepA were extracted from Swiss-Prot [14-16] and PDB databases [18]. Multiple sequence alignment of full length PepA from V. cholerae and E. coli and other species of V. cholerae was performed using clustal X [20, 21], with default parameters. Finally the multiple alignments were adjusted wherever necessary. Phylogenetic tree was constructed using Phylip Program [22]. 2.3. Model Building and Refinement Three dimensional homology model of PepA was built using crystal structure coordinates of PepA from E. coli (PDB ID: 1GYT) [18] as template. All steps of homology model building were carried out using program Modeller (Ver: 8v1) [19], this program is fully automated and constructs energy minimized protein models by satisfying restraints on bond distances and dihedral angles extracted from template PDB file. For PepA model in the presence of manganese, HETATM command of Modeller was employed and model was constructed using coordinates of PepA from E. coli [23]. Model was subjected to various cycles of Modeller [19] and best possible model was selected. For the reliability of the alignment, modeling of the variable surface loops and structural investigations was done using 3D visualization programs, Weblab Viewer [24, 25], DS Viewer [26] and Astex Viewer [27], in order to obtain more plausible model. 2.4. Model Evaluation An important part of the homology model building is the evaluation of the predicted model; this mainly involves the analysis of geometry distribution in the models. The reliability of the predicted structure was tested using the energy command of Modeller [19], PROCHECK [28, 29], web interface of WhatIF [30], Whatcheck [31] and ProSa [32]. In addition the variability in the predicted model, i.e., RMSD (Root mean square deviation) was calculated by superposition of C traces and backbones onto the template crystal structure [23]. Bonding interactions between active site residues and ligand was checked through running commands of Ligplot [33]. 3. RESULTS AND DISCUSSION 3.1. Sequence Comparision The pairwise sequence alignment for PepA from V. cholerae (SwissProt AC: P0C6E1) shows 80.9% sequence identity to PepA from E. coli (SwissProt AC: P68767) i.e., out of 503 amino acids 407 are identical (E-value: 1.0 E-4). The
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quality of alignment between the target and template sequences is the most important factor in the accuracy of the homology model. When the sequence identities are 50% or more, this becomes, more or less straightforward task, in this range of sequence homology the alignment is easier to construct. Few gaps exist and the structural differences between the proteins are usually limited to loops and side chains. The biochemical properties and primary sequence of PepA shows that it belongs to leucine aminopeptidase (LAP) family [11], M17 of metallopeptidases [8]. Analysis of approximately 6kb fragment in the vicinity of PepA gene revealed four open reading frames (ORFs). The organization of these four ORFs in V. cholerae is strikingly similar to that of E. coli. These ORFs encode a protein of 503 amino acids with a calculated Mol.wt of 54,617 Da and pI of 6.37. The amino acid sequence of PepA had a very high degree of similarity to PepA from E. coli [12], including perfect conservation of the residues believed to be involved in the active site of the enzyme, based on the crystallographic studies of PepA from E. coli [23]. Pairwise alignment shows that there is produced a gap at position 216 in the target sequence and at position 503 in the template (Fig. 1). There is no propeptide region in the sequence of PepA. In both these proteins 85% (1-165) of the residues are identical in N-terminal domain. The long chain - helix is 75% (166-192) conserved, 7 residues out of 20 are non-identical. 79% (193-503) identity is shown in the Cterminal region. Active site cleft is highly conserved. There is more N-terminal domain then C-terminal domain identity. Sequence comparison through multiple alignments among PepA from 15 different bacterial species including V. cholerae and E. coli shows high degree of conservation among members. Fig. (2) shows that high sequence conservation has been observed both at the C and N terminal regions, importantly at the catalytic site. Multiple sequence alignment revealed the presence of NTDAEGRL motif conserved in all the proteins, a conserved octapeptide which contains 2 residues involved in binding of metal ions, i.e. Asp351 and a Glu353, where as Arg355 being potentially catalytic amino acid. This is a characteristic signature of all known bacterial aminopeptidases. A phylogenetic tree has been constructed using multiple alignment of 15 aminopeptidase sequences from different bacterial species, results shows that PepA from both V. cholerae and E. coli are derived from common ancestors, but they are far from each other according to evolutionary point of view, it also shows that they belong to different orders (Fig. 3). 3.2. Homology Modeling of PepA Monomer The homology model of PepA monomer was obtained using 2.5 Ao resolution structure of PepA from E. coli [23] (PDB ID: 1GYT) [18] with which it shares 80.9% sequence identity. Studies have shown that PepA is a homohexamic protein like its template, i.e. it comprises of six identical chains, as it belongs to M17 family of LAPs which are hexameric and bind two cations [1, 34]. The E. coli PepA assembles in a homohexameric complex in which a monomer subunit with 503 amino acid residues comprises the unit
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Figure 1. Structure based target (PepA) - template (1GYT) alignment, showing high sequence conservation, identical residues are indicated by *, conserved active site residues (grey), potentially active residues (light grey), along with NTDAEGRL motif conservation are highlighted.
Figure 2. Multiple sequence alignment of 15 species of Aminopeptidase family, Aminopeptidase A both from V.cholerae (target) and E.coli (template) are shown in bold. Highly conserved residues are marked as*. Catalytically active residues are marked as $ while potentially catalytic residues are marked as @. The residues enclosed in box show NTDAEGRL motif conservation in all the species. (only the region showing the active site residues and the conserved motif in the multiply aligned sequences is shown here).
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-helices (a1, a4, a7 and a10) and 2 -sheets (b1, b2 antiparallel) from upper side whereas 6 -helices (a2, a3, a5, a6, a8 and a9) and 3 -sheets (b4, b8 parallel and b9 antiparallel) from lower side (Fig. 4C). Comparisons of the predicted model of V.choleare Pep A and X-ray crystallographic structure of E. coli PepA [34, 35], identified four loops (L1, L2, L3 and a disordered loop) in the N-terminal domains of these proteins (Fig. 4B). An extensive site-directed mutagenesis study confirmed the role of these loops in DNA binding and recombination [23]. These studies also implicated a more extended region of the PepA protein in site-directed recombination. Most notably, the aminopeptidase activity of PepA is not required for site-specific recombination [34]. The smaller N-terminal domain of PepA is proposed to play an important role in DNA binding [12, 23] like its template. It is also proposed that PepA may mediate pH regulation of virulence genes in V. cholerae by functioning as DNA binding protein and directly affecting transcription [12]. 3.3. Active Site & Proposed Enzyme Mechanism of PepA Model
Figure 3. Phylogenetic tree showing evolutionary relation ship between V. cholerae and other bacterial species. Target (dark grey) and template (light grey).
structure [12]. The overall topology and protein folding of the monomer model strongly resembles x-ray structure of E. coli, showing similar secondary structure pattern in both structures. The overall secondary structure composition shows 38.76% -helices, 20.47% -sheets, 28.42% random coils and 11.72 % turns. (Fig. 4A). Where as E. coli PepA has 40% helices and 20% sheets, showing close homology among the secondary structure elements. Monomer subunit comprises of two similar domain structure, smaller N-terminal domain (residue 1-165) is connected by a long chain - helix (residue 166-192), to the large Cterminal domain (residue 193-503). The helix has contacts with both C and N terminal domains. Both C and N terminal domains have mixed / structure; this has been inferred from the template 3D structure [23]. There is a disordered loop present both in the template and target structures from residue 146-152, but the residue 146 in target is Asn, which is replaced by Val in the template structure. These residues are involved in the refinement in order to facilitate the model interpretation and computational studies [23]. The core of both N and C terminal domain has a triple layered structure. A Six stranded -sheet (b1-b5 parallel, b6 anti parallel) is shielded by two -helices (a1 and a2) from one side, forms the core of N-terminal domain, the other side of this -sheet is shielded by 2 helices (a3 and a4) and loops (Fig. 4B). Where as the core of C-terminal domain has a triple layered structure consisting of a central eight stranded -sheets (b3, b5, b6, b7, b10, b11, b12 and b13), sandwiched between four
The predicted model of PepA shows that the catalytic activity of this enzyme is dependent on the presence of 2 Mn2+ ions. Each divalent metal ion is coordinated by 3 amino acids and a water molecule, because one amino acid residue binds with both metals. PepA with 2 metal ions have 5 amino acid metal ligands rather than 6, hence metal ions are pentahedrally coordinated Table 1. PepA is a metalloenzyme require Mn2+ for full activity and is inhibited by Zn2+, efficient cleavage of Leu-pnitroanilide substrate requires Mn2+. In E. coli PepA crystal structure 2 Zn2+ ions occupy the metal binding site which are occupied by Mn2+ in active enzyme [34]. The active site is located entirely with in the C-terminal domain, near the edge of central 8 -sheets. All the catalytically active residues are present in loops. (Fig. 5). There is present a bicarbonate ion in the crystal structure of the template bound to Arg356, which has a functional role in the mechanism of action of enzyme. Due to high degree of sequence and structure similarities between PepA from V. cholerae and PepA from E. coli, it can be predicted that if the target protein is crystallized in the same buffers as the template it may also binds this bicarbonate ion, which will bind to Arg355, same position as in the template [35] and it might function as a general base in enzyme mechanism of V. cholerae PepA same as in PepA from E. coli [35]. On the basis of crystallographic analysis Strater et al. suggested a potential mechanism of catalysis for PepA [1, 24, 34]. The active sites of PepA from E. coli (X-ray structure at 2.5 A resolution) and V. choleare (predicted model) are isostructural. On the basis of high sequence and structure homology between PepA from E. coli and V. choleare also other LAPs in the region of active site and metal ion binding environment it may be predicted that bicarbonate ion has critical importance in catalysis of V. choleare PepA as in the case with the analogous residues in E. coli PepA. There is now convincing evidence that this ion indeed has a functional role in the enzyme mechanism. PepA is significantly activated by physiological concentrations of bicarbonate, e.g., which are present in the cell from dissolved carbon di-
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Figure 4. Predicted monomer model of PepA. (A) Schematic representation of PepA showing the arrangement of secondary structure elements i.e. -helices, -sheets & coils. The N-terminal domain, C-terminal domain and domain linking helix. Also shown here are two manganese ions (dark grey). (B) N-terminal domain showing triple layered structure 5 -sheets sandwiched between 2 -helices on each side. Four loops (L1, L2, L3 and a disordered loop thought to be involved in DNA binding and recombination, There is present a disordered loop in the N-terminal region. (C) C-terminal domain showing central 8 -sheets sandwiched between 4 -helices & 2 -sheets from upper side while 6 -helices & 3 -sheets from lower side. Table 1.
PepA Residues (Target, Template) Implicated in Ion Coordination and Catalysis
Role
E. coli PepAa (Template)
V. choleare PepA b (target)
R Group Interactionc
Coordination of Mn 1 and Mn 2
Asp 275
Asp 274
Carboxylate oxygen
Glu 354
Glu 353
Carboxylate oxygen
Coordination of Mn1
Asp 352
Asp351
Carboxylate and carbonyl oxygen
Coordination of Mn2
Asp 293
Asp292
Carboxylate oxygen
Lys 270
Lys 269
Backbone amino group
Lys 282
Lys 281
Polarizes peptide carbonyl group
Arg 356
Arg 355
Binds bicarbonate
Catalysis
a
Active site residues were proposed based on X-ray crystal structures and residues in italics were tested for function using site-directed mutagenesis [34]. Active site residues were proposed based on sequence conservation. c R group interactions were proposed from crystal structures [34]. b
oxide. However, the presence of the bicarbonate ion is not essential for the proteolytic activity. Hydrogen bond donor groups from the guanidinium group of Arg-355 and a main chain NH group form a perfect binding pocket for the trigonal planar ion. A comparison to other metallopeptidases shows that these have proteinogenous carboxylate side chains that are proposed to serve as general bases in the enzyme mechanism (Fig. 6). The mechanism proposed by Strater et al. describes that, a bicarbonate anion is bound to an arginine side chain (Arg356 in E. coli PepA and Arg-355 in V. choleare PepA) very near to two catalytic Mn2+ ions. It is shown that PepA is ac-
tivated about 10-fold by bicarbonate when L-leucine-pnitroanilide is used as a substrate [35]. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a Mn-bridging water nucleophile to the peptide leaving group. The two Mn2+ ions are involved in the deprotonation of the nucleophile and together with Lys-281, in the polarization of the substrate carbonyl group and in transition-state stabilization.Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other metalloproteases peptidases. The proposed active-site residues are highly conserved in both target and template pro-
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Figure 5. Active site residues of Pep A; showing metal binding ligands (grey) and metal ions (Mn).
Figure 6. Active site structure and Proposed enzyme mechanism of PepA from E. coli & V. choleare, proposed by Strater et al. (A) Electron density map of Arg-355 (V. cholerae), (B) Electron density map of Arg-356 (E. coli). (C) Superposition of active site residues of target (residues highlighted in box) and template proteins. (D) The bicarbonate ion next to Arg-355(numbering according to PepA V. cholerae) acts as a general base and accepts proton from the metal-bridging water nucleophile. The proton is shuttled to the leaving peptide amino group to facilitate breakdown of the tetrahedral gem-diol intermediate. However, proton movements may not be unique. For example, water, molecules may be involved in the proton transfer paths. V. cholerae residues are shown in bracket.
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teins Table 1 and their importance has been tested using sitedirected mutagenesis for E. coli PepA [36]. 3.4. Quality of Proposed Model Using different validation tools quality of model was assessed. The homology models satisfy sterochemical restraints and passed all criteria implemented in Procheck [28, 29] and Whatcheck [31] to the same degrees as the template crystal structure refined at 2.5 Ao resolution. Ramachandran plot presents a good description of / angles of 503 amino acid residues of the predicted model obtained from Procheck. According to the results, there are 91.8% residues in most favored region (core), 7.7% in allowed region, 0.5% generously allowed region and there is no residue in disallowed region. From these results it can be inferred that PepA protein seems to be consistent with the representative energies of the crystal structure of PepA (1GYT). The structural superposition of C traces of PepA from V. cholerae and PepA from E. coli shown in Fig. (7). The RMSD between the template and target homology model calculated for C traces is 0.1740 Ao. Thus these values and the small variability between model and the template reflect the presence of strong restraints in most regions and emphasize very similar folding pattern among these enzymes. As expected from high sequence identity the two structures are well superimposed having similar folding topology but have different confirmation at certain regions. RMSD values between the two proteins point towards structural variability in few regions, despite high similarity in overall tertiary structure there are differences at position 143-
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148,213-217,283-284 and 503.Although there are differences in the target sequence, but these differences are in the loop regions, so it is reasonable to assume that it has little or no effect on overall conformation of the model. The active site residues are also well superposed with slight deviation at Lys 281 and Arg 355. The ligplot results show binding of amino acid to the metal ions. PepA with Mn2+ ions have 5 metal binding ligands, hence metal ions are pentahedrally coordinated. One metal is bonded by carboxylate of Asp274, Asp351 and Glu353 and the second by the amino group of Lys269 and the carboxylate of Asp292 and Glu353. Glu353 binds both metal ions and the backbone carbonyl group of Asp351 coordinates both metals (Fig. 8). A single water molecule bridges both metal ions. Lys282 is important for stabilization of substrate enzyme complex, which is known as gem-diolate [35]. Due to high similarity between the structure of template and target it can be predicted that Lys281 in target structure may also have important role in stabilization of enzyme substrate complex. The energetic architecture of protein folds was determined by using the program ProSA that gives the pair energy graph of PepA (Target) and PepA (Template) (Fig. 9). The energy graph of the predicted model and crystal structure were explored to examine the energy difference between them. Energy difference in region from 190-200 and 280-320 amino acid residues might be due to substitution of amino acid in this region. Energy difference in the region between residues 10–100 might be due to substitution of amino acids in this region. Z-
Figure 7. Structural superposition of C atoms of monomer homology model of PepA from V. cholerae (black) aligned with crystal structure of PepA from E. coli (grey), showing average RMSD value 0.174 between two structures, (i, ii, iii, iv, v, vi indicates differences in those regions); the active site residues are shown in ball & stick, they are also well superposed.
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Figure 8. LIGPLOT (A) E. coli (1gyt), (B) V. cholerae (PepA) showing the interaction between metal ions and metal binding ligands.
Figure 9. Pair Energy graph of PepA from V. cholerae (grey) and PepA from E. coli (black), using Protein Analysis Program (ProSa II), showing Z values slightly above 0 in few regions.
score was used as the measure of this energy, which indicated the quality of protein structures. Ideally the Z score should be below the zero point suggesting no significant stressed or strained folds with high energies. As is evident from the result, the energy graphs (Fig. 9) corresponded to highly stable structures and the values of energy, for the target molecule, slightly above zero point in some regions, which was in consistence with the values of experimentally found template crystal structure. That is why, from the energy stabilizations point of view, there seemed to be no problems in the modeled structure and on the basis of this graph, the modeled 3D structure can be acknowledged. 4. CONCLUSION PepA is a member of M17 family of leucyl aminopeptidases; its secondary structural elements show high conservation with the other members of the family. The M17 LAP monomers (53–55 kDa) assemble into homo-hexameric enzymes with high temperature (60–70oC) and alkaline pH (8.5–9.5) optima [34, 37, 38]. While efficiently hydrolyzing
peptide and amino acyl substrates with N-terminal Leu residues. It is well demonstrated that PepA from V. cholerae is closely related to PepA from E. coli, in terms of sequence and structural similarity and substrate specificity. As expected from high sequence identity PepA from V. cholerae is well superimposed over PepA from E. coli. PepA from V. cholerae is bi-lobed protomers that contain a larger Cterminal catalytic domain and a smaller, more variable Nterminal domain. PepA has two non-equivalent metalbinding sites (Mn1 and Mn2). Both ions have a role in substrate binding and catalysis, and are located at the edge of an eight stranded, saddle-shaped -sheet. It is observed that Mn2 is deeply imbedded in the PepA protomer and can exchanges ions slowly. In contrast, Mn1 is more accessible and can exchange readily. Manganese or magnesium ions are presumed to be present in the active LAPs, since these ions are potent activators of the animal, microbial and plant LAPs [1].
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The N-terminal domain of PepA from E. coli is known for DNA binding activity [23]. It has been proposed that PepA may mediate pH regulation of virulence genes in V. cholerae by functioning as DNA binding protein [12]. The N-terminal domain is reduced by 3 amino acid (1-163) in the V. cholerae which is 1-166 in template. The long stranded helix in target comprises of 28 residues, while the template has 25 residues. Metal ion is Zinc in crystal structure, while according to the literature this enzyme shows its full activity when Manganese is bound to it. Comparisons of the predicted model of V.choleare Pep A and X-ray crystallographic structure of E. coli PepA [34, 37], identified four loops (L1, L2, L3 and a disordered loop) in the N-terminal domains of these proteins. The similarity among both the enzymes led to the conclusion that PepA from V. cholerae will have same mechanism of action on substrate like its template. Also the predicted model can be helpful to study the structure activity relationship and enzyme substrate interactions. Vibrio cholerae PepA also regulates the expression of toxT-dependent virulence genes that control cholera toxin production [12, 39]. Acting as a DNA-binding protein, PepA represses toxT dependent genes in non-inducing pH conditions (pH 8.4).PepA N-terminal domain facilitates bending and wrapping of DNA, conferring its role as transcriptional repressor and mediator of recombination. The PepA domain or specific residues essential for mediating toxT repression are not currently known. Due to high degree of similarity between V. cholerae and E. coli PepA potent inhibitors can be designed for V. cholerae PepA on the basis of amino acids identified in E. coli PepA. LAPs are inhibited by chelators (EDTA and 1, 10phenanthroline).In addition, effective inhibitors of LAP includebestatin, amastatin, aminoaldehydes, L-leucinal, and leupeptin [37]. Bastatin and Amastatin are reported as inhibitors in template and in other aminopeptidases [3, 4]. It can be concluded that V. cholerae PepA may be inhibited by same inhibitors as that of E. coli PepA. Further studies will be done to dock these inhibitors into the V. cholerae PepA active site in order to analyze the binding of these inhibitors. Further more, homology modeling of hexamer; structure of V. cholerae PepA for studying the interaction across monomers and also for the study of DNA binding property would be interesting. ACKNOWLEDGMENTS Authors gratefully acknowledge Dr. Panjwani Centre of Medicine and Drug Discovery, International Centre for Chemical Sciences, Karachi (Pak) for provision of computational facilities and providing different bioinformatics softwares.
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Received: January 14, 2008
Revised: March 26, 2008
Accepted: June 16, 2008
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