Reactive cysteine in the active-site motif of Bacteroides thetaiotaomicron dipeptidyl peptidase III is a regulatory residue for enzyme activity

Abstract

Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide catabolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

“…The 100-kDa putative DPP3 (M r ϭ 102,897 in Val 2 -Glu 906 with vector tag M r ϭ 1416.5) exhibited activity with a preference for Arg at the P1 position and the highest activity for Arg-Arg-MCA (Fig. 2B), in accord with the properties of eukaryotic (36) and Bacteroides thetaiotaomicron DPP3 (37). Therefore, PGN_1645 truly represents P. gingivalis DPP3.…”

“…Therefore, PGN_1645 truly represents P. gingivalis DPP3. However, the observation that KDP136 did not hydrolyze Arg-Arg-MCA strongly suggested that DPP3 did not participate in extracellular dipeptide production in P. gingivalis and that it may be localized in the cytoplasm, as reported for B. thetaiotaomicron DPP3 (37).…”

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

“…Production of recombinant human DPP III was performed according to Š poljarić et al 28 . Gene amplification and expression of DPP III from Bacteroides thetaiotaomicron was described by Vukelić et al 16 . Protein purity was confirmed by SDS-PAGE according to Laemmli 29 .…”

“…These first two prokaryotic orthologs (from P. gingivalis and B. thetaiotaomicron) helped us to define five conserved regions in the peptidase family M49 14 . We have cloned and biochemically characterized the first bacterial DPP III, from the human gut symbiont B. thetaiotaomicron, revealing similarities but also important differences compared to its human counterpart 16 . Until now, crystal structures of two M49 family members have been solved, yeast and human DPP III 17,18 .…”

“…This is partly due to the lack of specific inhibitors. Inhibition by nonspecific chelating and thiol reagents is a characteristics of DPP III from various species 2,16,23 , but the studies of more specific inhibitors of the DPP III enzymatic activity are rare 24 . We have shown that two dipeptidyl hydroxamic acids, H-Tyr-Phe-NHOH and H-Tyr-Gly-NHOH act as the competitive inhibitors of human DPP III activity 20 .…”