Enhanced fructose oxidase activity in a galactose oxidase variant

被引：37

作者：

Deacon, SE ^{[1
]}

Mahmoud, K ^{[1
]}

Spooner, RK ^{[1
]}

Firbank, SJ ^{[1
]}

Knowles, PF ^{[1
]}

Phillips, SEV ^{[1
]}

McPherson, MJ ^{[1
]}

机构：

[1] Univ Leeds, Astbury Ctr Struct Mol Biol, Sch Biochem & Mol Biol, Leeds LS2 9JT, W Yorkshire, England

来源：

CHEMBIOCHEM | 2004年 / 5卷 / 07期

关键词：

copper enzymes; enzyme catalysis; fructose; oxiclases; protein engineering;

D O I：

10.1002/cbic.200300810

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：972 / 979

页数：8

共 28 条

[1]

AMARAL D, 1963, J BIOL CHEM, V238, P2281

[2]

AVIGAD G, 1962, J BIOL CHEM, V237, P2736

[3] ELECTRON-PARAMAGNETIC RESONANCE AND ELECTRON NUCLEAR DOUBLE-RESONANCE SPECTROSCOPIES OF THE RADICAL SITE IN GALACTOSE-OXIDASE AND OF THIOETHER-SUBSTITUTED PHENOL MODEL COMPOUNDS [J].