Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases*

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including l-carnosine and l-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepDCAT. The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an “extra” domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepDCAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepDCAT for GABAergic therapies or neuroprotection.

The substrates of PepD and carnosinases, L-carnosine and related Xaa-His dipeptides, are naturally occurring dipeptides involved in many biological pathways. Although the physiological role of L-carnosine is still uncertain, studies have indicated that L-carnosine exhibits a range of cytoprotective properties (26) that support its role as a cytosolic buffer (27), an antioxidant and free radical scavenger (28), and an antiglycation agent (29). Particularly, it is a potent and selective scavenger of ␣,␤unsaturated aldehydes and is known to inhibit aldehyde-induced protein-protein and DNA-protein cross-linking in neurodegenerative disorders such as Alzheimer disease, in cardiovascular ischemic damage, and in inflammatory diseases (30). Other roles ascribed to the small molecule include neuroprotective functions or action as neurotransmitters, modula-tion of enzymatic activities, formation of complexes with transition metals, regulation of macrophage function, and retardation of senescence in cultured fibroblasts (31,32). In particular, homocarnosine has been suggested as a precursor for the neurotransmitter GABA and acts as a GABA reservoir and may mediate the antiseizure effects of GABAergic therapies (33,34). Recently, Sauerhöfer et al. (35) also reported that glucose metabolism could be influenced by L-carnosine.
We have previously cloned and sequenced a biofilm-related and carnosine-hydrolyzing pepD gene from Vibrio alginolyticus (36,37). The V. alginolyticus PepD has a broader specificity for Xaa-His dipeptides and is sensitive to inhibition by bestatine and L-3,4dihydroxyphenylalanine (36,37). Bioinformatic analysis of the V. alginolyticus PepD protein revealed high sequence homology to that from other Vibrio species (94 -76% identity) and bacteria (75-63%). In contrast, sequence-based alignment of PepD with proteins from the metallopeptidase H Clan showed low sequence identities and similarities in the range of 7-20 and 13-34%, respectively (6 -8, 23, 36, 38 -40). Nevertheless, sequence analysis revealed that putative active site residues for catalysis are conserved in PepD and related di-zinc enzymes in the M20 family (6,23). His 80 , Asp 119 , Glu 150 , Asp 173 , and His 461 were predicted to be involved in metal binding, whereas Asp 82 and Glu 149 were predicted to be necessary for catalysis. Each of these residues were completely conserved, with the exception of Asp 173 . Asp 173 was present in homologs with aminopeptidase/dipeptidase specificity, although members of aminoacylase/carboxypeptidase contained a glutamic acid in the same position (36).
Several available crystal structures for the M20 family of enzymes, including PepV from Lactobacillus delbrueckii (6), CPG 2 from Pseudomonas sp. strain RS-16 (23), and CN2 from mice (14) have been reported. These enzymes have all exhibited an overall two-domain organization, a di-zinc binding catalytic domain, and a typical smaller domain. Nevertheless, CPG 2 and mouse CN2 are able to form homodimers, whereas PepV appears to exist only as a monomer (6,14,23). Although the biochemical activity of V. alginolyticus PepD has been well characterized, the structure-reaction mechanisms have not been studied in detail. Furthermore, little is known concerning the substrate specificity between L-carnosine and L-homocarnosine in PepD-catalyzed reactions.
We present herein the three-dimensional structure and putative substrate-binding residues of the V. alginolyticus PepD. We performed site-directed mutagenesis to determine which residues are involved in di-zinc metal ion binding and contribute to the architecture necessary for substrate C terminus binding and recognition. Furthermore, we found that the catalytic domain alone was sufficient for altering substrate specificity toward L-homocarnosine. Characterization of the PepD enzyme active-site architecture will aid in future studies to identify residues that may be modified to yield alternative substrate recognition properties and improve the potential therapeutic value of this protein and its closely related family members.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis of V. alginolyticus pepD Gene-Site-directed mutagenesis of the V. alginolyticus pepD gene was carried out using the Stratagene QuikChange site-directed mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA). Mutagenic primers were designed for use with the pET-28a(ϩ)-pepD plasmid (wild-type) template (as described below). Mutations were confirmed by DNA sequencing using the dideoxy chain termination method and the ABI PRISM 3100 autosequencer (Applied Biosystems, Foster City, CA). The recombinant mutant plasmids were transformed into E. coli BL21(DE3) pLysS competent cells for expression of the mutated PepD proteins.
Protein Expression and Purification-Both the PepD and PepD CAT proteins were produced and purified in the same manner. Colonies grown on an LB plate were inoculated into LB broth supplemented with 50 g/ml of kanamycin and grown at 37°C until A 600 of 0.5-0.6 was reached. At this point, protein production was induced by the addition of isopropyl thio-␤-Dgalactoside to a final concentration of 0.5 mM, and the culture was incubated at 37°C for an additional 4 h before harvest. The cells were collected by centrifugation and then resuspended in 15 ml of 20 mM Tris-HCl (pH 7.0) buffer containing 0.5 M NaCl. The mixture was sonicated, and the cell debris was removed by centrifugation at 11,000 ϫ g for 30 min at 4°C.
The supernatant containing recombinant proteins was loaded onto a Ni-Sepharose TM 6 Fast Flow column (GH Healthcare) previously prepared by washing with 10 column volumes of buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 7.0) containing 20 mM imidazole. The protein-loaded column was first washed with 5 column volumes of buffer A ϩ 20 mM imidazole, then with 5 column volumes of buffer A containing 70, 200, or 500 mM imidazole. Fractions of 1 ml each were collected, and the protein concentration in each fraction was determined using the Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific) with BSA as the standard. Fractions containing PepD enzymatic activity were pooled and dialyzed twice against 2 liters of 50 mM Tris-HCl (pH 7.0). The purified recombinant proteins were stored at Ϫ80°C for up to 6 months without loss of activity.
Enzymatic Activity Assay-The enzymatic activity was assayed according to the method described by Teufel et al. (13), which is based on the measurement of a histidine product by O-phthaldialdehyde (OPA) modification. Substrate hydrolysis was carried out in a final volume of 200 l containing 80 l of 50 mM Tris-HCl buffer (pH 7.0), 100 l of 2 mM L-carnosine (dissolved in 50 mM Tris-HCl, pH 7.0), and 20 l of purified enzyme (20 M). The reaction was initiated by the addition of substrate and terminated by the addition of 50 l of 1% trichloroacetic acid after a 30-min incubation at room temperature. Next, 50 l of 5 mg/ml of OPA dissolved in 2 M NaOH was added to derivatize the liberated histidine, and the reaction was incubated for 15 min at 37°C in darkness. The fluorescence of the OPAderivatized L-histidine was measured using Fluoroskan Ascent FL (Thermo Scientific, Waltham, MA) ( Exc , 355 nm and Em , 460 nm). Reactions with only L-histidine or only L-carnosine were treated in parallel to serve as positive and negative controls, respectively. All reactions were carried out in triplicate.
Enzyme Kinetics of PepD-For determination of V max , K m , and k cat of V. alginolyticus PepD wild-type and mutant proteins, the method described by Csámpai et al. (41) was slightly modified for use with HPLC and fluorescence detection. Different concentrations of substrate (0.25, 0.5, 1, 1.5, 2, 5, and 10 mM) were added to a nanomolar concentration of enzyme solution in 200 l at pH 7.4 for 20 min at 37°C. The liberated histidine was derivatized with 100 l of OPA reagent for 5 min at 37°C, and the fluorescence was detected as described previously. The substrate conversion did not exceed 20%. A total of nine substrate concentration points were used for each determination. The data collected were applied to the Lineweaver-Burk equation. The k cat /K m values reflect values assuming 100% activity of the enzyme preparation. All reactions were carried out in triplicate.
Substrate Specificity of PepD CAT -Various Xaa-His dipeptides (100 l, 2 mM) including ␤-Ala-L-His (L-carnosine), ␣-Ala-His, Gly-His, Val-His, Leu-His, Ile-His, Tyr-His, Ser-His, ␤-Asp-His, and ␥-aminobutyryl-His (GABA-His, L-homocarnosine), two His-Xaa dipeptides (His-Asp and His-Arg), and two histidine-containing tripeptides (Gly-Gly-His and Gly-His-Gly) were used to investigate the substrate specificity of PepD CAT . The reaction solutions were incubated for 25 min at 37°C and terminated by the addition of 50 l of OPA solution. Catalytic activity was analyzed spectrofluorimetrically, as described above. The activity measured for L-carnosine was defined as 100%.
Crystallization and Data Collection-Crystallization of PepD was performed at 291 K by the hanging-drop vapor-diffusion method against a reservoir solution containing PEG 400 (28%, v/v), 0.2 M CaCl 2 and 0.1 M Na-HEPES buffer (pH 7.5), as described previously (37). Crystals of a diamond shape appeared within six months and grew to maximum dimensions of 0.3 ϫ 0.2 ϫ 0.1 mm 3 . The protein crystals were transferred to the cryoprotectant solution containing glycerol (15%, v/v) prior to the x-ray diffraction experiment. Diffraction data were collected to 3.0-Å resolution on SPXF beamline BL13B1 at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan and beamline BL12B2 at SPring-8 in Japan. The data were processed using the HKL2000 suite (42). The redundancy independent merging R factor (R r.i.m. ) and the precision indicating merging R factor (R p.i.m. ) were calculated using the program RMERGE (43,44). The crystals belong to space group P6 5 with unit cell parameters a ϭ 80.42 Å and c ϭ 303.11 Å. The asymmetric unit contained two protein molecules, corresponding to a solvent content of 53.4%.
Structure Resolution and Refinement-The structure was solved by molecular replacement with MOLREP (CCP4) using the structure of Xaa-His dipeptidase from Haemophilus somnus 129PT (PDB code 2QYV) as the search model. The 2QYV was solved and deposited with the PDB by the Joint Center of Structure Genomics (JCSG), but was never published. The orientation of the lid domain was first located and fixed, subsequently leading to the determination of the relative position of the single catalytic domain. For structural refinement, the model was built using WinCOOT and refined using REFMAC5 (CCP4) to give the final R work ϭ 0.231 and R free ϭ 0.274, respectively (45). The Ramachandran results were determined using MOLPROBITY, and the percentage of residues in favored, allowed, and disallowed were 94.5, 98.6, and 1.4%, respectively (46). The structure found to have good stereochemistry was fully defined from Glu 3 to Glu 488 , with all main chain angles in the most favorable or generally allowed regions (47). All figures were produced using PyMOL. Data collection and refinement statistics are shown in Table 1.

RESULTS AND DISCUSSION
Overall Structure-The crystal structure of V. alginolyticus PepD was solved by the molecular replacement method and refined to a resolution of 3.0 Å with an R factor of 23.1% and an R free factor of 27.4% (Table 1). The overall structure of the PepD monomer is comprised of a total of 486 residues in two domains: an N-terminal catalytic domain harboring two zinc ions for catalysis and a C-terminal lid domain for substrate i.m. and R p.i.m. are as defined by Weiss (43).
where F o and F c are the observed and calculated structure factor amplitudes of reflection h. e R free is as R work , but calculated with 10% of randomly chosen reflection omitted from the refinement.
binding and protein dimerization (Fig. 1A). The x-ray absorption measurement and electron density map confirmed the presence and locations of Zn 2ϩ ions (supplemental Fig. S1). The high B-factors could be due to the flexible opened conformation between the catalytic and lid domains. PepD was also found to share similar structural folds with the PepV and related di-zinc-dependent M20/M28 family of enzymes, despite low sequence similarities among each. PepD and PepV showed root mean square deviations of 4.0 and 4.3 Å for C␣ atoms of the catalytic and lid domains, respectively. Two PepD molecules were found to be packed as a dimer in the asymmetric unit with dimensions of ϳ90 ϫ 90 ϫ 95 Å (Fig. 1B). The dimeric and monomeric characteristics of native and denatured PepD were also supported by evidence from analytical ultracentrifugation, which revealed molecular masses of 96.8 and 51.1 kDa under physiological and denatured conditions, respectively. The lid domain was found to utilize a hydrogenbonding network between helices from each monomer to form the dimer interface. PepD and related di-zinc-dependent enzymes of the M20/M28 family were determined to be dimers, whereas PepV was determined to exist as a monomer.
The Catalytic Domain-The topology of PepD and PepV is illustrated in Fig. 2. The PepD catalytic domain has a fold similar to that of PepV and the related di-zinc-dependent M20/M28 family of enzymes, including CPG 2 , ␤AS, mouse CN2, PepT, APAP, and SGAP (6,7,14,23,38,39). The catalytic domain consists of residues 3-186 and 401-488 and has mixed three-layer ␣/␤/ ␣-sandwich architecture with two ␤-sheet groups and seven ␣-helices (Fig. 3). The large sheet group contains eight strands arranged in the order a-b-f-c-g-j-h-i, in which b is the only antiparallel strand. The small sheet group is composed of four shorter antiparallel strands arranged in the order of d-e-l-k and located on the surface of the catalytic domain. The zinc ions are located at the C-terminal end of the four central parallel strands. The active site is located within a deep cleft formed between the lid and the catalytic domain (Fig. 1). Two active sites in the dimer are ϳ57 Å apart, suggesting that each protomer can function independently. No distinct zinc-bound water molecule was found; however, a higher electron density peak was observed with the closest zincwater contact of 2.5 Å. The absence of the zinc-bound water could be due to the limited resolution of the data. The N-and C-terminal ends are located on the top of the catalytic domain, opposite to the lid domain and the active site.
Biochemical studies of V. alginolyticus PepD revealed its metal-dependent characteristics (36) and optimal activation of apo-PepD has been observed with various divalent metal ions, including Mn 2ϩ , Co 2ϩ , Ni 2ϩ , Cu 2ϩ , and Cd 2ϩ (36). Previous studies have shown that the addition of Co 2ϩ ions to apo-PepD increases the enzymatic activity by a factor of ϳ1.4, compared with that of the wild-type PepD containing Zn 2ϩ . Moreover, Zn 2ϩ did not inhibit Co 2ϩ -loaded PepD activity. Substitution of Zn 2ϩ with Mg 2ϩ resulted in an approximate 80% restoration of the optimal enzymatic activity. In our study, the presence and locations of zinc atoms were confirmed by x-ray absorption and electron density map performed at beamline 13B1 (supplemental Fig. S1). The di-zinc center was found to be situated on the surface of the cleft between the catalytic and lid domains and, thus, is solvent-accessible. The crystal structure of PepD also revealed that several functional residues interact and fix two zinc ions (Zn1 and Zn2), which are separated by a distance of 2.8 Å (Fig. 4). Zn1 is coordinated by one of the carboxylate oxygens of Asp 119 , N⑀2 from His 461 , and a single putative water molecule bound by hydrogen bonding with the carboxylate group of Glu 149 . Zn2 is coordinated by N⑀2 of His 80 , the other carboxylate oxygen of Asp 119 , and two carboxylate oxygens of Asp 173 . Asp 119 is positioned as a bridging ligand between the two zinc ions. Notably, this residue is followed by an asparagine residue through a cis peptide bond as observed in many di-zincdependent enzymes of the M20/M28 family (23). This peptide bond is able to break the ␣-helix at the N-terminal end to position the Asp-Asn dipeptide closer in space than they would otherwise have been. An apparent difference in the orientation of the side chain carboxylate group of Glu 150 was noticed between the active-site center of PepD and that of the related M20/M28 family metallopeptidases. In PepV, the carboxylate oxygens of Glu 154 point inward to Zn1 with a distance of 1.9 and 2.6 Å, respectively; whereas in PepD the carboxylate oxygens of Glu 150 point away from the Zn1, and the distance between the carboxylate oxygen and Zn1 is 4.5 Å. Thus, the role of metal ion binding for Glu 150 in PepD remains ambiguous.
The Lid Domain-The lid domain of PepD consists of 214 residues (residues 187-400) between strands h and i of the ␤-sheet in the catalytic domain. It folds into a central eightstranded antiparallel ␤-sheet flanked on one side by four ␣-helices packed in alternating orientations (Fig. 5). The antiparallel ␤-sheets are arranged in the order of V-II-IV-III and VI-VII-I-VIII, respectively. Interestingly, the structure of the lid domain of PepD resembles that of PepV, but shares only a portion of the structure of related dimeric M20/M28 family enzymes, including CPG 2 , ␤AS, mouse CN2, and PepT. The CPG 2 dimer exhibits continuous ␤-sheets across the two monomers to form the dimer interface, whereas the lid domain of PepD formed the dimeric interface through hydrogen bonding between helices. Moreover, PepD formed a unique crisscross configuration via the interface interaction of the respective lid domains. Helices 6, 7, and 8 were found to participate in monomer-monomer contacts. Specifically, the carboxylate oxygens of Glu 294 and the hydroxyl group of Ser 374 , as well as the C ϭ O from the amide side chain of Asn 329 and the hydroxyl group of Ser 385 , are hydrogen-bonded to each other and form the dimeric interface (Fig. 6).
Structure Comparison of V. alginolyticus PepD and Related Di-zinc-dependent M20/M28 Family Enzymes-To further characterize the structural features of PepD, superimposition was carried out with the related M20/M28 metallopeptidases family members. The structure of PepD shows a close overall similarity to the uncharacterized PDB code 2QYV protein solved by the Joint Center for Structural Genomics (JCSG). The sequence alignment of these two proteins showed 50.9% sequence identity. The root mean square deviation of structure similarity between PepD and 2QYV for C␣ atoms was 0.63 and 0.73 Å among the catalytic and lid domains, respectively. Although both proteins share a structurally conserved active site, two notable regions in PepD connecting catalytic and lid domains (PepD residues 183-187 and 400 -403 versus code 2QYV residues 179 -183 and 397-400, respectively) showed minor differences in loop conformations between the proteins. In addition, the PepD protein also exhibited limited amino acid yet overall folding similarity to the M20/M28 metallopeptidases, except in the region of the dimer topology. The catalytic domain of PepD superpositioned well with the single domain structures of the Streptomyces griseus SGAP (39) and Aeromonas proteolytica APAP (38), in addition to the two domain structures of the Pseudomonas sp. CPG 2 (23), S. typhimurium PepT (7), Saccharomyces kluyveri ␤AS (8), human Acy1 (40), and mouse CN2 (14), as well as the counterpart of L. delbrueckii PepV (6). A major structural difference between PepD and the related di-zinc-dependent M20/M28 metallopeptidases is that the lid domain of PepD consists of an eight-stranded ␤-sheet and four ␣-helices similar to that of PepV, whereas the enzymes from the di-zinc-dependent M20/M28 family are composed of only one four-stranded antiparallel ␤-sheet flanked by two ␣-helices. Furthermore, part of the lid domain of the PepD structure is superimposed to that of the two domain structures of Pseudomonas sp. CPG 2 (23), S. typhimurium PepT (7), S. kluyveri ␤AS (8), human Acy1 (40), and mouse CN2 (14). These proteins are known to form a dimer interface through hydrophobic interactions between helices, as well as through hydrogen bonds between the two ␤-strands within the lid domain. Nevertheless, PepD exhibited a different dimeric architecture from that of the compared dimeric proteins in that the two lid domains of the dimeric proteins mediate enzyme dimerization through side by side packing of their four-stranded ␤-sheets to form contiguous extended eightstranded sheets. In contrast, a crisscross configuration was observed in PepD, wherein the lid domain formed the dimeric interface through hydrogen bonds between two sets of four ␣-helices (Fig. 1). Although the abovementioned M20/M28 family metallopeptidases all consist of homodimer structures similar to CPG 2 , no report in literature to date has discussed the PepD-like crisscross dimeric architecture. On the other hand, the structure of PepD aligns well with the counterpart of PepV (6), which is a monomer and does not have a known function in subunit dimerization. The lid domain of the PepV partially resembles the lid domain of CPG 2 but is about twice as large as that of CPG 2 . Moreover, the PepV lid domain extends itself away from the active site of the catalytic domain, folding over the active site and assisting the catalytic domain to form a cavity that is uniquely involved in substrate specificity (6). Surprisingly, the lid domain of PepD, which is also about twice as large as the lid domain of the CPG 2 and related dimeric proteins, is able to form a dimer instead of a monomer. Lindner et al. (40) reported that the eight-stranded ␤-sheets lid domain of PepV can be divided into two subdomains, both of which exhibit the same topology as the lid domain of CPG 2 and together can mimic the arrangement of the two lid domains within the CPG 2 and PepT dimers. However, dimerization of the subunits in PepD was mediated through hydrogen bonding of the ␣-helices instead of the side by side packing of the ␤-sheets. Moreover, one additional region (residues 186 -203 and 311-400) within the lid domain   Currently, the crystal structures of the M20 family of proteins have been reported for two different (open and closed) conformations. When the protein is crystallized in a free form, the catalytic and lid domains are in an orientation that exposes the active site to bulky water; whereas when the protein is complexed with an inhibitor, a closed conformation has been observed. In the PepV-inhibitor complex, a fixed "bridging" water molecule was found to be located between both zinc ions and close to the carboxylate group of the catalytic Glu 153 , which corresponds to Glu 149 of PepD and has been proposed as necessary for substrate hydrolysis (Fig. 7). Upon binding of the substrate, the water molecule will be positioned between the zinc ions and the carbonyl carbon of the scissile peptide bond.
Then, an attacking hydroxyl ion nucleophile is able to be subsequently generated through activation of the water molecule by both the zinc ions and transfer of the resultant proton to the Glu 153 . Proximal to the Glu 153 of PepV is the conserved metalbinding residue, Glu 154 , which utilizes its carboxylate oxygen to bind to the zinc ion with a distance of less than 3.0 Å. The carboxylate oxygen of Glu 154 of PepV is directed toward the Zn1. Nevertheless, our structural analysis of PepD in an open conformation revealed that the carboxylate oxygen of the corresponding Glu 150 residue of PepD is directed away from the Zn1 at a distance of 4.5 Å. It has been suggested that dipeptidases of M20 families can change their conformation from opened to closed during enzymatic catalysis (40). The conformational change could be achieved by a movement of the catalytic and lid domains. Consistent with this is the presence of a large clearance between the two domains that would allow a peptide chain to move to the opened active site, as was observed for the PepD structure. We, therefore, speculated that upon substrate binding, the PepD protein may change the metal ions' coordination and/or its protein conformation; the carboxylate oxygen of Glu 150 would be subsequently swung toward Zn1 and would push the Glu 149 -bound water molecule toward Zn2, effectively bridging the water between the two zinc ions. However, the precise molecular interactions between the enzyme active site and the substrate or inhibitor still await final x-ray structure determination.
The di-zinc binding ligands, His 80 , Asp 119 , Glu 150 , and His 461 , are conserved among all of the proteins compared in this study, but the Asp 173 was found to be replaced by Glu in CPG 2 and hACy1. This finding is consistent with the observation reported by Lindner et al. (40) that all homologs with proven aminopeptidase or dipeptidase specificity contain an aspartic acid, whereas a glutamic acid residue has been identified in the same position in Acyl1/M20 family members that exhibit either aminoacylase or carboxypeptidase specificity. In   addition, four additional residues were found to be conserved among all of the compared proteins: 1) Asp 82 , which is two residues downstream from the His 80 in the vicinity of the zinc center and is assumed to clamp the imidazolium ring of His 80 ; 2) Glu 149 , a putative general base for enzyme catalysis; and 3) His 219 and Arg 369 , putative substrate C-terminal and/or transition state binding residues. On the other hand, within the PepV, CPG 2 , and related M20/M28 family metallopeptidases, a cispeptide bond exists between the bridging Asp and the proximal residue. In CPG 2 , APAP, PepT, and PepV, this residue is an Asp, which is replaced by Asn in PepD (Asn 120 ) and SGAP (Asn 98 ). The cis-peptide has been proposed to have a function in forcing the bridging carboxylate to conform to the correct geometry for metal binding (23).
Mutational Analysis on Metal-binding and Catalytic Residues of V. alginolyticus PepD-Previously, His 80 , Asp 119 , Glu 150 , Asp 173 , and His 461 were described as being putatively involved in metal binding in PepD. We individually mutated each of these residues using alanine-scanning mutagenesis and characterized the expressed proteins with CD spectrometry (supplemental Fig. S2). Each of the mutations produced similar quantities of the protein and exhibited homologous CD spectra. Nevertheless, no activity could be detected, indicating the essential role of these residues in the enzymatic activity of PepD (Tables 2 and 3). In a parallel experimental procedure, Asp 119 was substituted with Glu, Met, Leu, Ile, Arg, Phe, Ala, Ser, Thr, Cys, Pro or Asn, Glu 150 was replaced with Arg or His, and Asp 173 was mutated to a Glu residue. As expected, substitution of Asp 119 with other proteinogenic amino acid residues completely abolished enzymatic activity. On the other hand, substitution of Glu 150 with Asp led to the retention of ϳ60% of the maximal hydrolytic activity of the wild-type enzyme, whereas substitution of Glu 150 with Arg or His completely abolished enzymatic activity. Substitution of Asp 173 with Glu also completely abolished the enzymatic activity.
Next the Asp 82 and Glu 149 residues, which are both putatively involved in catalysis, were subjected to site-directed mutagenesis. Asp 82 was substituted for Gly, Val, Phe, Tyr, His, or Glu, whereas the Glu 149 was replaced with Gly, Ala, Ile, Ser, His, Trp, or Asp. No activity was detected for any of the Asp 82 mutants. Substituting Glu 149 of PepD with Gly, Ala, Ile, Ser, His, Trp, or Asp also resulted in the abolishment of enzymatic activity, except for the Asp mutant, which retained ϳ55% of the wild-type activity (Tables 2 and 3). It is interesting to note that replacement of Glu 149 or Glu 150 with aspartic acid led to partial retention of enzymatic activity despite being only one carbon shorter and having the same negative charge. We speculate that shortening the amino acid side chain in this position may allow for its acidic group to move away from an optimum position to promote activation of the catalytic water molecule, or perhaps the replacement of Glu with Asp at this position may partially affect the metal ligand-binding affinity and subsequent activation of the catalytic water for substrate-enzyme tetrahedral intermediate formation. This may have, in turn, resulted in partial loss of the enzymatic activity.
To investigate whether Asn 120 of the cis-peptide is involved in catalysis or protein folding/stabilization, Asn 120 was substituted with Ala and its enzymatic activity was examined. As expected, the activity was not detected in the PepD N120A mutant. In addition, the CD spectra of the PepD wild-type and PepD N120A mutant proteins presented almost the same shape in the range of 198 -250 nm (supplemental Fig. S2), implying that the PepD N120A mutant protein was not perturbed in either its stability or folding properties. These results indicate that Asn 120 plays an essential role in the enzyme reaction.

Mutational Analysis on Probable Substrate C-terminal Binding Residues within the Lid Domain of V. alginolyticus PepD-
Jozic et al. (6) have previously identified three residues, Asn 217 , His 269 , and Arg 350 , within the lid domain of PepV that are putatively involved in the substrate C-terminal and/or transition state binding through hydrogen bonding. Due to the different topology of the ␤-sheet order, a simple primary sequence alignment was not able to identify the corresponding residues in the lid domain of PepD, except for Arg 369 , which aligned with Arg 350 of PepV. This residue also superimposed with Arg 324 , Arg 280 , and Arg 276 in the small domains of the dimeric CPG 2 , PepT, and hACy1, respectively. We then used structure-based sequence alignment to identify the other equivalent residues in PepD. A structure superimposition but inversed sequence order, in which the Asn 217 and His 269 residues of PepV superimposed with the Asn 260 and His 219 residues of PepD, was noticed. Asn 260 is conserved among PepV, CPG 2 , ␤AS, and  PepT, but is substituted by Thr in human CN1 and mouse CN2 as well as by Tyr in PepT. Remarkably, the His 219 , Asn 260 , and Arg 369 residues are located on the same side of the lid domain for both PepD dimers, but the corresponding residues are located on the opposite side of the lid domain of the same monomer for CPG 2 and related dimeric proteins (Fig. 5). Therefore, in CPG 2 , the Arg 288 from the lid domain of one monomer interacts with the His 229 and Asn 275 from the lid domain of the other monomer; in contrast, the Arg 369 from the lid domain of the PepD monomer interacts with the Asn 260 and His 219 from the lid domain of the same monomer.
We also performed site-directed mutagenesis experiments to test the roles of these equivalent lid domain residues. The mutated PepD proteins were produced in a procedure similar to that of the wild-type PepD. All mutants exhibited similar purification characteristics and the same electrophoretic mobility as the wild-type enzyme in SDS-PAGE. Although each of the mutations produced similar quantities of the protein, the Arg 369 to Ala mutation resulted in complete loss of the enzymatic activity for hydrolyzing L-carnosine, whereas the Asn 260 to Ala mutation decreased the catalytic activity to almost half. Interestingly, the His 219 to Ala mutation did not affect the enzymatic activity significantly, yielding only a slight increase in activity of ϳ10% as compared with the wild-type PepD. In PepV, the Arg 350 was located near the C terminus of the bound inhibitor (2.7 Å) but appeared to be too far away from the zinc ions (ϳ8 Å), indicating a role in substrate binding but not in catalysis. The replacement of Arg with Ala might disrupt the hydrogen bond network between the Arg 369 side chain and Asn 260 N␦ with the carboxylate group of the substrate. In the case of PepV, Jozic et al. (6) have argued that domain flexibility is required to allow substrate access. Moreover, the bad diffraction and high mosaicity observed in the inhibitor-free PepV crystal have been attributed to conformational variability between open and closed states. A significant opening of the protein conformation would clearly benefit access of the peptides to the active site cavity. It is conceivable that even the whole lid domain might move away from its site to allow for easier substrate access and product egress. Therefore, although the Arg 369 guanidinium side chain and the Asn 260 N␦ within the active site of PepD are both ϳ16 Å away from the zinc ion, a conformational change between the open and closed states might have contributed to the movement of both Arg 369 and Asn 260 upon substrate binding and subsequent transition state stabilization. Furthermore, binding of the His 219 in PepD to the substrate likely persists during the conformational change between the open and closed states and contributes to transition-state stabilization through an electrostatic interaction between His 219 and the free carboxyl group of the ligand, as shown in the PepV-inhibitor complex (Fig. 7).
Mutational Analysis on Dimeric Interface of V. alginolyticus PepD-Based on the crystal structure, PepD and PepV revealed similar architecture, except that PepD is a dimer and PepV is a monomer. From the structural illustration of PepD, the dimeric interface appears to be formed by hydrogen bonds through Ser 374 and Ser 385 of one subunit to Glu 294 and Asn 329 of the other subunit, respectively (Fig. 6). We then performed site-directed mutagenesis experiments on these residues to investigate the putative dimeric interface interactions. According to the results obtained from analytical sedimentation velocity ultracentrifugation, the molecular mass of the PepD S374A/S385A double mutant was determined to be 54.4 Ϯ 0.02 kDa, whereas the PepD wild-type was 96.8 Ϯ 0.11 kDa. Size exclusion chromatography of PepD WT and the PepD S374A/S385A double mutant also revealed the corresponding sizes of ϳ103.7 and 50.6 kDa, respectively (supplemental Fig. S3). These results suggested that the PepD S374A/S385A mutant existed as a monomer in solution. Interestingly, the PepD S374A/S385A mutant exhibited ϳ130% activity of the wild-type, indicating independent function for the monomer. However, the exact reason for forming the dimeric structure remains unclear, but a physiochemical or regulatory function of PepD may be involved.
Substrate Specificity Alteration of the Truncated PepD Catalytic Domain-We have previously observed that the catalytic domain of PepD contains metal binding sites and is responsible for substrate hydrolysis, whereas the lid domain plays a role in substrate recognition. To further substantiate the functional role of the catalytic domain, we constructed a truncated enzyme of the PepD catalytic domain alone (PepD CAT ) and investigated the preference for substrate specificity. The catalytic domain regions, comprised of residues 1-186 and 401-490, were PCR amplified, ligated, and subcloned into the pET28a(ϩ) vector to construct the PepD CAT recombinant plasmid. The truncated protein was produced similar to the wildtype PepD and exhibited the expected size of ϳ31 kDa. The substrate specificity of PepD CAT was determined at pH 7.4 and 37°C with 14 peptides, including 10 Xaa-His dipeptides, two His-Xaa dipeptides, and two His-containing tripeptides (Table  4). Compared with the enzymatic activity of wild-type PepD, the activity of PepD CAT was significantly reduced or not measurable, except for L-carnosine or L-homocarnosine. The Pep-D CAT exhibited ϳ20% of the wild-type activity toward the L-carnosine substrate. Unexpectedly, the PepD CAT protein exhibited altered substrate specificity to L-homocarnosineas compared with that of the full-length PepD protein, and with ϳ6% of the activity. The results suggested that the substrate selectivity of the PepD CAT protein hold the potential for appli- cation in GABAergic therapies or as a neuroprotector, because L-homocarnosine is a precursor for GABA and acts as a GABA reservoir.
Proposed Catalytic Mechanism-The structural similarity between PepD and related M20/M28 family metallopeptidases led to the hypothesis that these enzymes share a common catalytic mechanism. Based on superimposition of putative metaland substrate-binding residues among these enzymes, a general mechanism may be described that: (a) before substrate binding, a bridging water molecule is positioned between two Zn 2ϩ ions and spatially close to the carboxylate group of the catalytic Glu 149 ; (b) the PepD undergoes a conformational change upon substrate binding and hydrolysis; (c) during catalysis, the catalytic Glu 149 acts as a general base by promoting the nucleophilic attack of the metal-bound water on the substrate carbonyl carbon and transfers the proton to the Glu 149 ; (d) the carbonyl oxygen then binds in an "oxyanion binding hole" formed by Zn1 and the imidazole group of His 219 in PepD, resulting in polar-ization of the carbonyl group and facilitating the nucleophilic attack of the scissile bond by the zinc-oriented hydroxyl group; (e) this leads to a tetrahedral intermediate, which subsequently decays to the product after one additional proton transfer from the catalytic Glu 149 carboxylate to the amide nitrogen in His 219 . In addition to the catalytic Glu 149 , mutational analyses indicated that putative substrate binding residues Asp 82 , Glu 149 , and Arg 369 play essential roles in the hydrolysis reaction (Fig. 8).
In summary, despite the lack of detectable sequence homology, the PepD has clear structural homology to other di-zinc-dependent M20 and M28 family of enzymes. The structure of V. alginolyticus PepD reveals it to be a dimer with two domains in each subunit. The catalytic domain of PepD contains two zinc ions and is structurally homologous to other proteolytic enzymes with dinuclear zinc catalytic sites. The lid domain of PepD is structurally homologous to that of PepV, but with different topology of ␤-sheet order. Interestingly, part of the lid domain of the PepD structure is also homologous to the lid domain of the dimeric proteins. Nevertheless, PepV exists as a monomer, whereas the PepD and related di-zinc-dependent M20/M28 family of enzymes were determined to be dimers. Structural comparisons between PepD and related di-zinc-dependent metallopeptidases suggest that formation of the catalytically competent active site in the PepD family of enzymes may be associated with transition from an open to a closed enzyme conformation. In parallel, the site-directed mutation of the putative substrate C-terminal binding residues, D260A and R369A, resulted in complete loss or partial decrease of the enzymatic activity. Furthermore, the enzymatic assay of the truncated PepD catalytic domain, PepD CAT , further demonstrated the functional role of the lid domain in substrate binding and selectivity. Finally, the structural data on PepD reported here may inspire strategies for the improvement of the PepD family of enzymes toward applications in biotechnology and allow the design of targeted disease peptidases or prodrugs with altered specificity.