Chapter 3 Results
3.8. Structure features of Vibrio alginolyticus PepD
To predict the active site residues of V. alginolyticus PepD, the computational analysis was also carried out through homology modeling analysis. The PepD model was obtained with L. delbrueckii PepV as the template, and the generated PepD structure is quite similar to PepV structure (Fig. 18).
. The PepD catalytic domain has a fold similar ons at llow) including e quite similar.
ology modeling, seven residues including His80, Asp1
ought to be involved in the active site of V. alginolyticus PepD crucial for associating with inc ions , and Asp82 and Glu149, two catalytic residues. Moreover, the superimposition of Fig. 18 Three-dimensional ribbon of the crystal structure of PepV(right) and the generated PepD(left) model based on PepV
to the dinuclear carnosine-hydrolyzing enzyme PepV. The structure predicted two zinc i the catalytic pocket center which were held by five metal binding residues (ye
the adjacent residue Asp119 (light green). The residues for catalytic (blue) wer
Apparently, based on the results of sequence alignment, activity assay, and hom 19, Glu150, Asp119, Asp173, His461 were th
z
PepV active site residues with PepD revealed that the predicted active site residues of PepD were almost equivalent to that of PepV (Fig. 19).
H461 (PepD)
ig. 19 Stereo view of PepD (blue) superimposed with the active site residues of PepV.
The
gray sticks are residues of PepV, and the orange sticks (metal binding) and the blue sticks (catalytic) are residues of PepD. The predicted metal binding residues of PepD are almost equivalent to that of PepV. The yellow stick is the phosphinic inhibitor AspΨ [PO2CH2]AlaOH of PepV.
It was considered that the active site residues of enzymes in M20 family were almost conserved. Carboxypeptidase G2 (CPG2), the first dinuclear dipeptidase with available crystal structure in M20 family, was proven to cleave the the C-terminal Glu from folic acid and folate analogs.58 The structure homology models showed that the catalytic domain of PepV has a similar folding to the CPG2 and the active sites residues of PepV were fully conserved in CPG2.55 In the other hand, the sequence of PepD showed 19.3% identity and 34.2%
similarity with CPG2. To determine the putative catalytic domain of PepD, the sequence alignment and superimposition of CPG2 active site residues with PepD was also performed.
The result revealed that the predicted active site residues of PepD were almost equivalent to that of PepV and CPG2 (Fig. 20, 21), only the residue Asp119 of PepD displayed a notable difference in this model. However, loss of enzymatic activity of PepD D119 mutant indicated that this residue might play an important role for the PepD mediated cleavaging reaction.
D119(PepD)
Fig. 20 Local view of PepD superimposed with the active site residues of PepV and CPG2. The residues were respectively colored in gray, yellow and blue for PepD, PepV and CPG2. The active site residues were almost equivalent. The zinc binding residue Asp173 in PepD and equivalent aspartic acid in PepV was substituted by glutamic acid in CPG2.
Proposed active site residues : Asp82 ,Glu149
Proposed metal ion binding residues : His80, Asp119,Glu150, Asp173, His461 Fig. 21 Multiple sequence alignment with CPG , PepV and PepD 2
3.9 Crystallization of Vibrio alginolyticus PepD
The V. alginolyticus PepD crystals were obtained by using hanging drop technique as described in 2.12. Hanging drops were formed by mixing 1 μL of 10 mg/mL enzyme solution with 1 μL crystallization reagent. The crystals were yielded at 20 ºC in two weeks under two condition: Crystallization reagent was composed of (a) 20% PEG-4000, 10% isopropanol, 0.1 M Na-HEPES, pH 7.5 (b) 28% PEG-400, 0.1 M Na-HEPES, 0.2 M CaCl2, pH 7.5 (Fig.
22). The crystals diffraction was 2.7 Å at best, but the quality of this data collection was not useful for phasing. Therefore, further modification of the precipitation condition should be undertaken and hope to obtain the high-resolution crystals for structure determination in the future.
(b) (a)
Fig. 22 The V. alginolyticus PepD crystals were grown at 20 ºC in two condition: (a) 20%
8% PEG-400, 0.1 M a-HEPES, 0.2 M CaCl2, pH 7.5
PEG-4000, 10% isopropanol, 0.1 M Na-HEPES, pH 7.5 (b) 2 N
Chapter 4 Discussion and conclusion
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.
Chapter 5 Future work
Based on the results in this study, two putative active site residues which were apparently involved in the catalysis of PepD were first identified. The saturated mutagenesis on other putative metal binding and catalytic sites will be continuous analyzed to clarify their roles in the peptidase activity of PepD. Moreover, several residues outside of the catalytic domain of enzymes in M20 family were considered to be involved in the substrate binding and catalysis in recent report. As described on section 1.9, the respective His269 from the lid domain of L.
elbrueckii PepV was associated with Zn 1 for forming an oxyanion binding hole bound to e carbonyl oxygen of Glu 153 and led to a tetrahedral intermediate. This residue functioned r the stabilization of the transition state and its corresponding results to His229, His223, and His206 in the small domains of the dimeric homologs CPG2, PepT, and hAcy1 in M20 family were also be examined. The mutagenesis study on H206 of hAcyl in 200326 indicated at the conserved histidine in the dimerization domain of dimeric Acy1/M20 family
zymes contributes in trans to the active site. Therefore, several polar or aromatic residues outside of the putative active site of PepD should also be futher investigated by site-directed mutagenesis in the future.
PepD was identified as a member of metallopeptidases which required metal ion for its catalytic activity. In these peptidases, the metal ion is usually zinc but sometimes cobalt, manganese, nickel or copper. However, recent studies showed that different metal ion with rious concentration could inhibit or increase the enzyme activity.42, 64 The functional roles r the different metal centers as well as the activation mechanism due to lose metal ions are still unclear. Consequently, the metal selectivity and inhibition/activation mechanism will be valuable investigated in the future.
d th fo
th en
va fo
As the biological function of bacterial pepD is less understanding, the gene knock-out study on V. alginolyticus pepD followed by a series of biochemical or morphology analysis will provide more informations about its role in prokaryotes. At the same time, since PepD affects the bacterial biofilm formation, biofilm assay should also be performed and compared with both V. alginolyticus wild-type and pepD knockout strain.
With absence of the structure in neither peptidase family M20 nor similar peptidase, crystallization on the V. alginolyticus PepD was needed. Furthermore, the crystal structure of the wild-type and mutant proteins combined with the mutagenesis analysis data could provide an insight into the catalytic mechanism of bacterial aminoacylhistidine dipeptidase.
Up to date, we have obtained the crystal of wild-type PepD with undesirable resolution.
Therefore, the proceeding effort is still needed to modify the crystallization condition in order to improve the quality of crystal for further structure determination.
Cha
obiol. 1961, 5, ( ), 477-86 . Sakazaki, R., Proposal of Vibrio alginolyticus for the biotype 2 of Vibrio parah
nd infections. J lin Microbiol 1975, 2, (6 ), 556-8.
7. Baross, J.; Liston, J., Occurrence of Vibrio parahaemolyticus and related hemolytic vibrios in marine environments of Washington State. Appl Microbiol 1970, 20, (2), 179-86.
. Paperna, I., Reproduction cycle and tolerance to temperature and salinity of myloodinium ocellatum (Brown, 1931) (Dinoflagellida). Ann Parasitol Hum Comp 1984, 59, (1 ), 7-30.
9. Zen-Yoji, H.; Le Clair, R. A.; Ota, K.; Montague, T. S., Comparison of Vibrio arahaemolyticus cultures isolated in the United States with those isolated in Japan. J Infect
is 1973, 127, (3 ), 237-41.
10. Levine, W. C.; Griffin, P. M., Vibrio infections on the Gulf Coast: results of first year of regional surveillance. Gulf Coast Vibrio Working Group. J Infect Dis 1993, 167, (2 ), 479-83.
Reina Prieto, J.; Hervas Palazon, J., [Otitis media due to Vibrio alginolyticus: the risks of the Mediterranean Sea]. An Esp Pediatr 1993, 39, (4 ), 361-3.
pter 6 Reference
1. Goarant, C.; Merien, F.; Berthe, F.; Mermoud, I.; Perolat, P., Arbitrarily primed PCR to type Vibrio spp. pathogenic for shrimp. Appl Environ Microbiol 1999, 65, (3 ), 1145-51.
2. Miyamoto, Y., Nakamura, K., and Takizawa, K., Proposals of a new genus
“Oceanomonas” and of the amended species names. Jpn. J. Micr 3
aemolyticus. Jpn J Med Sci Biol 1968, 21, (5 ), 359-62.
4. Schmidt, U.; Chmel, H.; Cobbs, C., Vibrio alginolyticus infections in humans. J Clin Microbiol 1979, 10, (5 ), 666-8.
5. Molitoris, E.; Joseph, S. W.; Krichevsky, M. I.; Sindhuhardja, W.; Colwell, R. R., Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia. Appl Environ Microbiol 1985, 50, (6 ), 1388-94.
6. Rubin, S. J.; Tilton, R. C., Isolation of Vibrio alginolyticus from wou C
12. Gahrn-Hansen, B.; Hornstrup, M. K., [Extraintestinal infections caused by Vibrio
2002, 15, (4 ), 757-70.
ibrio alginolyticus in the grouper, Epinephelus labaricus, Bloch et Schneider
Res 2006, 34, (Database issue ), D270-2.
Chem Rev 1996, 96, (7), 2435-2458.
2003, 7, parahaemolyticus and Vibrio alginolyticus at the county of Funen 1987-1992]. Ugeskr Laeger 1994, 156, (37), 5279-82.
13. Rippey, S. R., Infectious diseases associated with molluscan shellfish consumption. Clin Microbiol Rev 1994, 7, (4), 419-25.
14. Blake, P. A.; Weaver, R. E.; Hollis, D. G., Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol 1980, 34, ( ), 341-67.
15. Lipp, E. K.; Huq, A.; Colwell, R. R., Effects of global climate on infectious disease: the cholera model. Clin Microbiol Rev
16. Caccemese SM, R. D., Chronic diarrhea associated with Vibrio alginolyticus in an immunocompromised patient. Clin Infect Dis. 1999, 29, ( ), 946-47.
17. Lessner, A. M.; Webb, R. M.; Rabin, B., Vibrio alginolyticus conjunctivitis. First reported case. Arch Ophthalmol 1985, 103, (2 ), 229-30.
18. Lee, K. K., Pathogenesis studies on V
ma . Microb Pathog 1995, 19, (1 ), 39-48.
19. Rawlings, N. D.; Barrett, A. J., Evolutionary families of metallopeptidases. Methods Enzymol 1995, 248, ( ), 183-228.
20. Rawlings, N. D.; Morton, F. R.; Barrett, A. J., MEROPS: the peptidase database.
Nucleic Acids
21. Rawlings, N. D.; Barrett, A. J., Evolutionary families of peptidases. Biochem J 1993, 290 ( Pt 1), ( ), 205-18.
22. Wilcox, D. E., Binuclear Metallohydrolases.
23. Dismukes, G. C., Manganese Enzymes with Binuclear Active Sites. Chem Rev 1996, 96, (7), 2909-2926.
24. Richard C Holz, Krzysztof P Bzymek and Sabina I Swierczek Co-catalytic metallopeptidases as pharmaceutical targets Current Opinion in Chemical Biology
(2 ), 197-206.
25. Wouters, M. A.; Husain, A., Changes in zinc ligation promote remodeling of the active site in the zinc hydrolase superfamily. J Mol Biol 2001, 314, (5 ), 1191-207.
erization for catalysis in the aminoacylase-1/M20
Martin, J.; Stribbling, S. M.;
stander efficacy in two xenograft
li biosynthesis regulator
t 10 ), 2847-57.
specificity. FEMS Microbiol Lett 1994, 123, (1-2 ), 153-9.
hir 1980, 35, (17 ), 1283-6.
teriol
Dembinski, D. R.; Hartman, P. E.; Miller, C. G., Salmonella typhimurium 26. Lindner, H. A.; Lunin, V. V.; Alary, A.; Hecker, R.; Cygler, M.; Menard, R., Essential roles of zinc ligation and enzyme dim
family. J Biol Chem 2003, 278, (45 ), 44496-504.
27. Friedlos, F.; Davies, L.; Scanlon, I.; Ogilvie, L. M.;
Spooner, R. A.; Niculescu-Duvaz, I.; Marais, R.; Springer, C. J., Three new prodrugs for suicide gene therapy using carboxypeptidase G2 elicit by
models. Cancer Res 2002, 62, (6), 1724-9.
28. Brombacher, E.; Dorel, C.; Zehnder, A. J.; Landini, P., The cur
CsgD co-ordinates the expression of both positive and negative determinants for biofilm formation in Escherichia coli. Microbiology 2003, 149, (P
29. Klein, J.; Henrich, B.; Plapp, R., Cloning and expression of the pepD gene of Escherichia coli. J Gen Microbiol 1986, 132, (8 ), 2337-43.
30. Schroeder, U.; Henrich, B.; Fink, J.; Plapp, R., Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate
31. van der Drift, C.; Ketelaars, H. C., Carnosinase: its presence in Pseudomonas aeruginosa.
Antonie Van Leeuwenhoek 1974, 40, (3 ), 377-84.
32. Bersani, M.; Crespi, F.; Giudici, V.; Bianchi, G., [Antral stenosis caused by gastric hemangioma]. Minerva C
33. Miller, C. G.; Schwartz, G., Peptidase-deficient mutants of Escherichia coli. J Bac 1978, 135, (2 ), 603-11.
34. Kirsh, M.;
peptidase active on carnosine. J Bacteriol 1978, 134, (2 ), 361-74.
35. Jackson, M. C.; Lenney, J. F., The distribution of carnosine and related dipeptides in rat
and human tissues. Inflamm Res 1996, 45, (3 ), 132-5.
tioxidant activity of
P.; Borin, G., Carnosine and carnosine-related
2001, 56, (6 ),
arand, S.; Carreau, A.; Cairns, N.
ction from cellular carnosinase, and
ood, R. A.; Amir, A.; Idowu, B.; Summers, B.; Leigh, N.; Peters, T.
36. Boldyrev, A. A., Problems and perspectives in studying the biological role of carnosine.
Biochemistry (Mosc) 2000, 65, (7 ), 751-6.
37. Vaughan-Jones, R. D.; Spitzer, K. W.; Swietach, P., Spatial aspects of intracellular pH regulation in heart muscle. Prog Biophys Mol Biol 2006, 90, (1-3 ), 207-24.
37. Vaughan-Jones, R. D.; Spitzer, K. W.; Swietach, P., Spatial aspects of intracellular pH regulation in heart muscle. Prog Biophys Mol Biol 2006, 90, (1-3 ), 207-24.