Functional analysis of two serine peptidases from Treponema putidum

Abstract

Objectives: To establish the biochemical activities of two putative serine peptidases (MEROPS S9-family) encoded by Treponema putidum. Methods: Putative prolyl oligopeptidase (Tput-POP; JO40_13405; 682aa) and putative prolyl iminopeptidase (Tput-PIP; JO40_03260; 313aa) proteins encoded by T. putdum OMZ-758T were cloned, expressed and purified as N-terminal hexahistidine-fusions. Their abilities to hydrolyze chromogenic/fluorogenic peptide substrates including: L-Pro-pNA, H-Gly-Ala-Pro-AFC, H-Gly-Pro- βNA, Suc-Ala-Ala-Pro-Ala-pNA, N-Suc-Ala-Ala-Pro-Phe-pNA (SAAPFpNA), were quantified spectrophotometrically/fluorometrically (Spectramax multi-wavelength plate-reader) at 37°C, pH 7.4 [βNA=2-naphylamine; AFC=7-amino-4-(trifluoromethyl); pNA= p-nitroanilide]. The activities of T. denticola PIP (TDE2776) and POP (TDE1195) were assayed in parallel. Results: The monomeric Tput-POP protein efficiently hydrolyzed Z-Gly-Ala-Pro-AFC with a molar specific activity of 498±11 micromoles/min.micromoles protein; slightly higher than that of TDE1195. Suc-Ala-Ala-Pro-Ala-pNA and L-Pro-pNA were also hydrolyzed, albeit with low efficiencies. The Tput-PIP protein hydrolyzed L-Pro-pNA more efficiently than TDE2776; with molar specific activities of 98±10 and 21±3 micromoles/min.micromoles protein, respectively. Tput-PIP also digested SAAPFpNA with modest activities (0.74±0.02 micromoles/min.micromoles protein), whilst TDE2776 had negligible activities against this substrate. Conclusions: These preliminary results indicate that T. putidum PIP and POP serine peptidases have activities similar to the corresponding homologues from T. denticola. This suggests that T. putidum and T. denticola utilize similar peptidase systems to degrade proline-containing peptide fragments, e.g. those produced via proteolytic degradation of host-derived tissues, such as collagen.