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PEDS Advance Access originally published online on January 12, 2004
Protein Engineering Design and Selection 2004 17(2):141-148; doi:10.1093/protein/gzh018
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© 2004 Oxford University Press

Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution

D. Wilkinson1, N. Akumanyi, R. Hurtado-Guerrero, H. Dawkes2, P.F. Knowles, S.E.V. Phillips and M.J. McPherson3

Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 1Present address: Delta Biotechnology Ltd, Castle Court, 59 Castle Boulevard, Nottingham NG7 1FD, UK 2Present address: Biologics SRC, Pfizer Ltd, Sittingbourne, Kent ME9 8AG, UK

3 To whom correspondence should be addressed. e-mail: m.j.mcpherson{at}leeds.ac.uk

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high KM for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower KM than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved KM in the C383S variant protein.

Received September 19, 2003; revised December 19, 2003; accepted December 24, 2003 Edited by Alan Fersht


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 Protein Eng Des SelHome page
J. Paramesvaran, E. G. Hibbert, A. J. Russell, and P. A. Dalby
Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies
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