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PEDS Advance Access originally published online on December 20, 2005
Protein Engineering Design and Selection 2006 19(2):77-84; doi:10.1093/protein/gzj004
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis

C. Madzak1, M.C. Mimmi2, E. Caminade3, A. Brault3, S. Baumberger2, P. Briozzo2, C. Mougin3 and C. Jolivalt4,5

1UMR Microbiologie et Génétique Moléculaire, INRA/CNRS/INA-PG, CBAI and 2UMR INRA-INAPG 206 de Chimie Biologique, F-78850 Thiverval-Grignon, 3Unité de Phytopharmacie et Médiateurs Chimiques, INRA, route de Saint-Cyr, F-78026 Versailles Cedex and 4Laboratoire de Synthèse Sélective Organique et Produits Naturels, UMR CNRS 7573, ENSCP, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France

5 To whom correspondence should be addressed. E-mail: claude-jolivalt{at}enscp.fr

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for kcat between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH ({Delta}pH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Keywords: fungal laccase/pH-dependence/polyphenol oxidase/site directed mutagenesis/Yarrowia lipolytica expression system

Received July 14, 2005; revised November 25, 2005; accepted December 2, 2005.

Edited by Jacques Fastrez


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