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PEDS Advance Access published online on January 12, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh018
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Received September 19, 2003
Revised December 19, 2003
Accepted December 24, 2003

Article

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

D Wilkinson 1, N. Akumanyi 2, R. Hurtado-Guerrero 2, H. Dawkes 3, P.F. Knowles 2, S.E.V. Phillips 2, and M.J. McPherson 2*

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

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


  Abstract

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper-containing enzyme that oxidises 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 characterised a number of single, double and triple mutants at residues Cys 383, Tyr 436 and Val 494, identified in an earlier directed evolution study, 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 3d 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.

Keywords: copper oxidase/directed evolution/galactose oxidase/guar gum/ substrate binding


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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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