title: Biochemical characterization of recombinant β-carbonic anhydrase (PgiCAb) identified in the genome of the oral pathogenic bacterium Porphyromonas gingivalis.
abstract: Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ-, δ- and ζ-CAs are ubiquitous metalloenzymes present in prokaryotes and eukaryotes. CAs started to be investigated in detail only recently in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many such organisms they are essential for the life cycle of the organism. CA inhibition leads to growth impairment or growth defects in several pathogenic bacteria. The microbiota of the human oral mucosa consists of a myriad of bacterial species, Porphyromonas gingivalis being one of them and the major pathogen responsible for the development of chronic periodontitis. The genome of P. gingivalis encodes for a β- and a γ-CAs. Recently, our group purified the recombinant γ-CA (named PgiCA) which was shown to possess a significant catalytic activity for the reaction that converts CO2 to bicarbonate and protons, with a kcat of 4.1 × 10(5 )s(-1) and a kcat/Km of 5.4 × 10(7 )M(-1 )× s(-1). We have also investigated its inhibition profile with a range of inorganic anions such as thiocyanate, cyanide, azide, hydrogen sulfide, sulfamate and trithiocarbonate. Here, we describe the cloning, purification and kinetic parameters of the other class of CA identified in the genome of P. gingivalis, the β-CA, named PgiCAb. This enzyme has a good catalytic activity, with a kcat of 2.8 × 10(5 )s(-1) and a kcat/Km of 1.5 × 10(7 )M(-1 )× s(-1). PgiCAb was also inhibited by the clinically used sulfonamide acetazolamide, with an inhibition constant of 214 nM. The role of CAs as possible virulence factors of P. gingivalis is poorly understood at the moment but their good catalytic activity and the fact that they might be inhibited by a large number of compounds, which may pave the way for finding inhibitors with antibacterial activity that may elucidate these phenomena and lead to novel antibiotics.
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR , Napoli , Italy .
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.forename: Viviana
authors.initials: V
authors.lastname: De Luca
authors.forename: Zeid
authors.initials: Z
authors.lastname: AlOthman
authors.forename: Sameh M
authors.initials: SM
authors.lastname: Osman
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
mesh_terms: D000086:Acetazolamide / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D011994:Recombinant Proteins; D013449:Sulfonamides; D002256:Carbonic Anhydrases; D000086:Acetazolamide
keywords: Carbonic anhydrase; PgiCAb; Porphyromonas gingivalis; pathogenic bacteria