title: Laboratory-evolved vanadium chloroperoxidase exhibits 100-fold higher halogenating activity at alkaline pH: catalytic effects from first and second coordination sphere mutations.
abstract: Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.
authors.affiliation: Van't Hoff Institute of Molecular Sciences, University of Amsterdam, 1018 WS Amsterdam, The Netherlands.
authors.forename: Zulfiqar
authors.initials: Z
authors.lastname: Hasan
authors.forename: Rokus
authors.initials: R
authors.lastname: Renirie
authors.forename: Richard
authors.initials: R
authors.lastname: Kerkman
authors.forename: Harald J
authors.initials: HJ
authors.lastname: Ruijssenaars
authors.forename: Aloysius F
authors.initials: AF
authors.lastname: Hartog
authors.forename: Ron
authors.initials: R
authors.lastname: Wever
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D044646:Ascophyllum / Q000201:enzymology; D001966:Bromine / Q000378:metabolism; D002384:Catalysis; D002711:Chloride Peroxidase / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D006219:Halogens / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction
chemical_list: D006219:Halogens; C418625:vanadium chloroperoxidase; D002711:Chloride Peroxidase; D001966:Bromine