title: 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.
authors.affiliation: Division of Organic Chemistry and Biochemistry, Rudjer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia.
authors.forename: Bojana
authors.initials: B
authors.lastname: Vukelić
authors.forename: Branka
authors.initials: B
authors.lastname: Salopek-Sondi
authors.forename: Jasminka
authors.initials: J
authors.lastname: Špoljarić
authors.forename: Igor
authors.initials: I
authors.lastname: Sabljić
authors.forename: Nevenka
authors.initials: N
authors.lastname: Meštrović
authors.forename: Dejan
authors.initials: D
authors.lastname: Agić
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
mesh_terms: D020816:Amino Acid Motifs; D001439:Bacteroides / Q000201:enzymology*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013696:Temperature
chemical_list: D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D003545:Cysteine