title: Molecular cloning and biochemical characterization of Xaa-Pro dipeptidyl-peptidase from Streptococcus mutans and its inhibition by anti-human DPP IV drugs.
abstract: Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg(-1), while the Vmax and Km were 7 μM min(-1) and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents.
authors.affiliation: School of Pharmacy, University of Camerino, Camerino, Italy.
authors.forename: Arpan
authors.initials: A
authors.lastname: De
authors.affiliation: School of Pharmacy, University of Camerino, Camerino, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Lupidi
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy.
authors.forename: Dezemona
authors.initials: D
authors.lastname: Petrelli
authors.affiliation: School of Pharmacy, University of Camerino, Camerino, Italy luca.vitali@unicam.it.
authors.forename: Luca A
authors.initials: LA
authors.lastname: Vitali
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004151:Dipeptides / Q000494:pharmacology; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D054873:Dipeptidyl-Peptidase IV Inhibitors / Q000494:pharmacology*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013295:Streptococcus mutans / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D004151:Dipeptides; D054873:Dipeptidyl-Peptidase IV Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; C041445:diprotin A; C502994:saxagliptin; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4; D000218:Adamantane
keywords: Serine protease; enzyme inhibition; gliptins; nisin induction; protein purification