title: Modification of halogen specificity of a vanadium-dependent bromoperoxidase.
abstract: The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3). In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.
authors.affiliation: Department of Biotechnology, Tottori University, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohshiro
authors.forename: Jennifer
authors.initials: J
authors.lastname: Littlechild
authors.forename: Esther
authors.initials: E
authors.lastname: Garcia-Rodriguez
authors.forename: Michail N
authors.initials: MN
authors.lastname: Isupov
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Iida
authors.forename: Takushi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Izumi
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006219:Halogens / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D014639:Vanadium / Q000378:metabolism*
chemical_list: D006219:Halogens; D011994:Recombinant Proteins; D014639:Vanadium; D010544:Peroxidases; C026195:bromide peroxidase