Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.
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 K(M) 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 K(M) 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 K(M) in the C383S variant protein.
Wilkinson
D
D
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Akumanyi
N
N
Hurtado-Guerrero
R
R
Dawkes
H
H
Knowles
P F
PF
Phillips
S E V
SE
McPherson
M J
MJ
D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine; D019020:Directed Molecular Evolution / Q000379:methods*; D005692:Galactose Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D005692:Galactose Oxidase; D003545:Cysteine