title: Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.
abstract: Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA from Vibrio cholerae (VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 × 10(5) s(-1) and a kcat/Km of 4.1 × 10(7) M(-1) s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Ki of 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.
authors.affiliation: Dipartimento di Chimica `Ugo Shiff', Polo Scientifico, Università degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino, Firenze, 50019, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Ferraroni
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.affiliation: Dipartimento di Chimica `Ugo Shiff', Polo Scientifico, Università degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino, Firenze, 50019, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy.
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
authors.affiliation: Dipartimento di Chimica `Ugo Shiff', Polo Scientifico, Università degli Studi di Firenze, Via della Lastruccia 3, Sesto Fiorentino, Firenze, 50019, Italy.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
mesh_terms: D000086:Acetazolamide; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001639:Bicarbonates / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014734:Vibrio cholerae / Q000737:chemistry* / Q000201:enzymology; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D021122:Protein Subunits; D011522:Protons; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D024402:Carbonic Anhydrase II; D015032:Zinc; D000086:Acetazolamide
keywords: Vibrio cholerae; carbonic anhydrase; crystal structure; type II CA; β-carbonic anhydrase