title: Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).
abstract: The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.
authors.affiliation: E. C. Slater Institute, Faculty of Chemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Hemrika
authors.forename: R
authors.initials: R
authors.lastname: Renirie
authors.forename: S
authors.initials: S
authors.lastname: Macedo-Ribeiro
authors.forename: A
authors.initials: A
authors.lastname: Messerschmidt
authors.forename: R
authors.initials: R
authors.lastname: Wever
mesh_terms: D020134:Catalytic Domain; D002711:Chloride Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship; D014639:Vanadium
chemical_list: D011994:Recombinant Proteins; D014639:Vanadium; D002711:Chloride Peroxidase