Enhanced Fructose Oxidase Activity in a Galactose Oxidase Variant

Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously,...

Full description

Saved in:
Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2004-07, Vol.5 (7), p.972-979
Main Authors: Deacon, Sarah E, Mahmoud, Khaled, Spooner, R. Kate, Firbank, Susan J, Knowles, Peter F, Phillips, Simon E.V, McPherson, Michael J
Format: Article
Language:English
Subjects:
copper enzymes
Crystallography, X-Ray
enzyme catalysis
fructose
Fructose - metabolism
Galactose - metabolism
Galactose Oxidase - chemistry
Galactose Oxidase - genetics
Galactose Oxidase - metabolism
Mutation
oxidases
Oxidation-Reduction
Pichia - genetics
protein engineering
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Structure-Activity Relationship
Substrate Specificity
Transformation, Genetic
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200300810