title: The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae.
abstract: The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252-residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26-residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an alpha-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and II suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active-site residues are identical to those found in the high-activity human isozyme II. The bacterial enzyme has a high CO2 hydration activity with a k(cat) of 1.1 x 10(6) s(-1) and Km of 20 mM at pH 9 and 25 degrees C. The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pKa of 6.7 and a maximal value of the catalytic second-order rate constant, k(enz), of 130 M(-1) x s(-1).
authors.affiliation: Department of Biochemistry, Umeå University, Sweden.
authors.forename: L C
authors.initials: LC
authors.lastname: Chirică
authors.forename: B
authors.initials: B
authors.lastname: Elleby
authors.forename: B H
authors.initials: BH
authors.lastname: Jonsson
authors.forename: S
authors.initials: S
authors.lastname: Lindskog
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D002256:Carbonic Anhydrases