Atas, Ali (2014) Functional analysis of Oligopeptidase A from 'Yersinia pseudotuberculosis'. (MSc(R) thesis), Kingston University, .

Abstract

Proteases have long been studied and analyzed. However, some are more well known than others. Oligopeptidase A (OpdA) is an example of an enzyme that requires particular attention, as preliminary results by Karlyshev ‘et al’ (unpublished results) has demonstrated its absence in ‘Yersinia pseudotuberculosis’ results in a reduction of virulence when introduced into mice. Over-expression, purification and enzymatic activity of OpdA from ‘Y. pseudotuberculosis’ is reported in this study. OpdA-His was successfully over expressed and purified with a molecular weight of 77 kDa. The enzyme displayed no activity against resorufin labeled casein but was able to cleave the short fluorogenic substrates; Abz-NKPRRPQEDDnp and Abz-AAL-EDDnp. The hydrolysis of Abz-NKPRRPQ-EDDnp and Abz- AAL-EDDnp by OpdA-His was enhanced in the presence of Ca2+, C02+ and Mn2+. Interestingly, OpdA-His was inhibited by 0.1 mM and 1 mM Zn2+ and Cu2+, and stimulated by 0.001 mM and 0.01 mM Zn2+. Metal chelating agent EGTA at 1 mM concentration was unable to fully inhibit the hydrolysis of Abz-NKPRRPQ-EDDnp but fully inhibited the hydrolysis of Abz-AAL-EDDnp after 5 minutes. Likewise, the inhibitory effect of 0.1 mM chymostatin, 0.02 M N-[N-(N-Acetyl-L-Ieucyl)-L-Ieucyl]-L-norleucine (ALLN) and 74IJM antipain was more dramatic on the hydrolysis of Abz-AAL-EDDnp than the hydrolysis of Abz-NKPRRPQ-EDDnp. In order to determine the ability of OpdA-His to cleave products of other proteases and give an insight into the role of OpdA-His, several attempts were made to purify the ATP-dependent protease Lon. Unfortunately, these attempts were unsuccessful and Lon was not purified. Glycine extracts of ‘Escherichia coli’ cells over-expressing OpdA-His did not present any bands on SDS-PAGE. Furthermore, to have an understanding whether OpdA-His was involved in antimicrobial peptide breakdown, E. coli cells over-expressing OpdA-His were incubated with different concentrations bradykinin for 24 hr at 37 degrees Celsius. Interestingly, over-expression of OpdA-His reduced cell viability. Overall, these results give strong evidence that OpdA from ‘Y. pseudotuberculosis’ is a Zn-dependent metallo-protease that harbors a concentration dependent inhibitory zinc-binding site.

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