In this study, V. alginolyticus PepD which identified as a member of metallopeptidase M20 family and considered as an aminoacylhistidine dipeptidase was being investigated. The recombinant PepD of V. alginolyticus with a His-tag on N inus was successfully expressed and purified with high purity. Based on the SDS-PAGE analysis, the molecular mass of PepD is about 55 kDa near to the calculated molecular mass of 53.6 kDa. and is also similar to that of previously identified E. coli PepD.29
-term
which was similar to that exhibited by all of the known PepD. It could hydrolyze the unique dipeptide L-carnosine (β-Ala-L-His), which has potential neuroprotective function in brain and could act as an antioxidant or antiglycation agent. The substrate specificity of PepD in V. alginolyticus was also identified in this study. It could degrade a large number of Xaa-His dipeptides besides brain-specific dipeptide like GABA-His. The amino group in α or β position of N-terminus residue did not affect the recognition and hydrolysis of dipeptide. However, with the more tendency for α-Ala-L-His than L-carnosine as substrate was first identified in bacterial PepD, whereas the same result could also be observed in human cytosolic nonspecific dipeptidase (CN2).42 Several Xaa-His dipeptides including Val-His, Leu-His, Tyr-His, Ile-His, His-His and Ser-His were superior over carnosine as substrates for degradation and the enzyme also could hydrolyze Gly-His ith good activity but had no apparent activity on β-Asp-L-His. Therefore, we preliminary
part or C-terminal position were not degraded, indicating at V. alginolyticus PepD is a Xaa-His dipeptidase in activity. The study of substrate
ecificity on Xaa-His dipeptides was first carried out in bacterial aminoacylhistidine
The V. alginolyticus PepD enzymatic activity was performed on
L-carnosine-hydrolyzing,
w
assumed that this enzymatic activity on Xaa-His dipeptides is dependent on the charge of Xaa amino acid. The His-Xaa dipeptides including His-Ile or His-Val, as well as tripeptides containing histidine in the central
th sp
dipeptidase, but had already been identified in mammals.
-1 -1
-1 -1), PepD catalyzes at a relative low efficiency. However, the Km value of V. alginolyticus PepD was lower than PepD of
Escherichia coli 30
alginolyticus
The kinetic values including kcat or kcat/Km, were compared with the mammal aminoacylhistidine dipeptidases while lacking of the related reports for the bacterial PepD and PepV. The Km value and catalytic efficiency (kcat/Km) of V. alginolyticus PepD for
L-carnosine were 0.36 mM and 0.398 mM s , respectively. As compared with that of human carnosinase (CN1) (Km 1.2 mM and kcat/Km 8.6 mM s
K-12 (2 to 5 mM) indicated a relatively higher interaction of V.
PepD with its substrates. Based on the result of higher hydrolysis rate on α-Ala-L-His than β-Ala-L-His as the substrate, PepD could have other more suitable dipeptide substrate for degradation or even another totally different enzymatic activity.
Moreover, the inhibitiory effect of bestatin on L-carnosine hydrolysis by PepD will be investigated.
According to the result of sequence alignment between PepV and PepD, the active site residues were almost conserved. We predicted that there are five putative metal binding residues, His80, Asp119, Glu 150, Asp173, and His461, and two catalytic residues, Asp82 and Glu149 in the enzymatic active site cavity. The conservation of the active site residues suggests that the hydrolytic mechanism of PepD and PepV might be closely related. The PepD mutants created by site-directed mutagenesis on putative metal binding residue Asp119 resulting in losing the activities but without changing the secondary structure with CD spectra analysis. It revealed that this residue might be involved in the metal binding for the dramatically affecting enzymatic activity. There were no detectable zinc ions in the D119E mutant crystal by X-ray diffraction, preliminary confirming our hypothesis. We also investigated the residue of catalytic Glu149 for site-directed mutagenesis. Surprisingly,
almost the mutants lost the activities besides E149D that retained partial enzymatic activity.
The similar spectra and the predict percentages of secondary structure from CD spectra compared with the wild-type PepD via CD spectra analysis confirmed that the similar secondary structure was existed in the mutant E149D. Based on this result, we suggested that changing of the putative catalytic residue glutamic acid to the aspartic acid with the same negative charge would keep the enzymatic ability to interact with the substrate.
To confirm our suggestion, the orientation relationship between the putative active site residues and zinc ions were investigated by molecular modeling. The carbonyl oxygen of the mutant D119E was far away from zinc ion to involve in the metal binding ability for the dramatically affecting enzymatic activity (Fig. 23).
Fig. 23 Stereo view of orientation relationship between zinc ion and the putative metal binding residues Asp119.
The catalytic mechanism proposed that the bridging catalytic water attacks the carbonyl carbon of the scissile peptide bond to form a sp3-orbital substrate-enzyme tetrahedral intermediate (Fig.24). The distant from the catalytic water to the carbonyl carbon of the mutant E149D was too far to from the substrate-enzyme tetrahedral intermediate that further involved in substrate binding ability and caused the mutant losing partial enzyme activity (Fig. 25).
Fig. 24 Proposed catalytic mechanism for the hydrolysis of N-terminal amino acid residues. Proposed general mechanism for the hydrolysis of a peptide, catalyzed by a
inal carboxylate.
metallopeptidase with a co-catalytic active site where R1, R2, R3 are substrate side chains and R is an N-terminal amine or a C-term
R4
Fig. 25 Stereo view of orientation relationship between catalytic water and the putative catalytic residues Glu149.
The homology modeling structure of PepD was obtained from the L. delbrueckii PepV crystal structure with dizinc nuclear was quite similar. The putative residues for catalytic Asp82 and Glu149 of PepD were primarily superimposed on PepV Asp89 and Glu153 residues. Asp82 was conserved in all of the active enzymes from clan MH and considered to
p the imidazolium ring of His80. Glu149 served as a general base in catalysis, whereas
water molecule. Moreover, the position of the metal binding residues of PepD, PepV and CPG2 were almost clam
the water molecule was bridged by two zinc ion acting as the attacking hydroxyl ion nucleophile.58 These two zinc ions, as described by Jozic et al.,55 were considered to play two different roles for hydrolyzing substrates: for stabilization of the substrate-enzyme tetrahedral intermediate as well as for activation of the catalytic
superim
indistinguishable except for Asp119 (Asp141) which was considered as the bridging residue held both two zinc ions in PepV (CPG2). The orientation of Asp119 in PepD seems with less association for two zincs. However, mutants with losing activity provide some evidences for Asp119 involving in the catalytic reaction undoubtedly. Thus, we assumed that the active site pocket of PepD and PepV were similar and the hydrolytic mechanism might also be closely related but with slight difference.
Enzymes with the known crystal structures in M20 family such as PepV was identified as monomer whereas CPG2 as homodimer in their native state. The native form of V.
alginolyticus PepD was analyzed on the Native-PACE as well as western blotting analysis.
Based on the Native-PAGE and the film, we assumed that both wild-type and mutant PepD existed in several forms while monomer form was formed in dominant. However, the result of analytical sedimentation velocity ultracentrifugation indicated that there were only
formed and non-covalent interaction was apparently weak. The imerized PepD might be separated by electricity through electrophoresis analysis.
homodimer in the native state of PepD .It is possible that none of the covalent interaction between PepD proteins was
d
In conclusion, V. alginolyticus PepD was considered as a member of aminoacylhistidine dipeptidase which could hydrolyze Xaa-His dipeptides including an unusual dipeptide carnosine (β-Ala-L-His) with low catalytic efficiency. The further investigation on substrate specificity indicated that V. alginolyticus PepD was considered to be a Xaa-His dipeptidase that hydrolyze various His-containing dipeptides except the dipeptide with the negative charge in its N-terminal part. V. alginolyticus PepD is similar to the CN2 that could not hydrolyze the brain-specific dipeptidases such as GABA-His, but different from the PepV in losing the degradation ability toward unusual tripeptides. In native state, PepD existed in several forms but preferred to form homodimer. Mutagenesis study and
homology modeling structure on PepD revealed that the putative active site pocket of PepD might be similar to PepV, even the hydrolytic mechanism was closely related but with slight different. As a member of peptidase family M20, the most direct evidence on the metal content of PepD is determined through the progressive crystallization study for characterizing the mono- or di-zinc catalytic center. Either the actual active site pocket and hydrolytic mechanism will also be characterized via the crystallography.