title: Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA.
abstract: Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. CAs are present in many pathogenic species and are involved in the bicarbonate metabolism/biosynthetic reactions involving this ion. Ubiquity of these enzymes suggests a pivotal role in microbial virulence and pathogenicity. Porphyromonas gingivalis is an anaerobic bacterium, which colonizes the oral cavity, being involved in the pathogenesis of periodontitis, an inflammatory disease leading to tooth loss. Recently, we reported an anion inhibitory study on the γ-CA (denominated PgiCA) identified in the genome of this Gram-negative bacterium. In this paper we continue our research on PgiCA, and describe the biochemical characterization of the recombinant protein, its thermal stability, the oligomeric state and the enzyme kinetics. PgiCA is a polypeptide chain formed of 192 amino acids and displays an identity of 30-33% when compared with the prototypical γ-CAs, CAM or CAMH (from Methanosarcina thermophila) or CcmM (from Thermosynechococcus elongatus). A subunit molecular mass of 21 kDa was estimated by SDS-PAGE, while HPLC size exclusion chromatography under native conditions gave an estimated molecular mass of 65 kDa suggesting that the recombinant enzyme self-associate in a homotrimer, as all other γ-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other γ-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.
authors.affiliation: Istituto di Biochimica delle Proteine , CNR, Napoli , Italy .
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.forename: Viviana
authors.initials: V
authors.lastname: De Luca
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carginale
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002256:Carbonic Anhydrases
keywords: CO2 hydration; enzyme kinetic; metalloenzymes; periodontitis; γ-carbonic anhydrase