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J. Biol. Chem., Vol. 281, Issue 14, 9738-9744, April 7, 2006

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Laboratory-evolved Vanadium Chloroperoxidase Exhibits 100-Fold Higher Halogenating Activity at Alkaline pH

CATALYTIC EFFECTS FROM FIRST AND SECOND COORDINATION SPHERE MUTATIONS^*

Zulfiqar Hasan, Rokus Renirie, Richard Kerkman, Harald J. Ruijssenaars, Aloysius F. Hartog, and Ron Wever¹

From the Van't Hoff Institute of Molecular Sciences, University of Amsterdam, 1018 WS Amsterdam, The Netherlands and Dutch States Mines Anti-Infectives B.V., Delft, The Netherlands

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 H₂O₂ (16 µM) 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.

Received for publication, November 11, 2005 , and in revised form, January 17, 2006.

^* This work was supported by the Netherlands Organization for Scientific Research (NWO), The Netherlands Technology Foundation (STW), the Dutch National Research School Combination (NRSC-Catalysis), and DSM Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Van't Hoff Institute of Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands. Tel.: 31-20-5255110; Fax: 31-20-5255670; E-mail: rwever{at}science.uva.nl.

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				J. M. Winter and B. S. Moore Exploring the Chemistry and Biology of Vanadium-dependent Haloperoxidases J. Biol. Chem., July 10, 2009; 284(28): 18577 - 18581. [Abstract] [Full Text] [PDF]