1T2X

Glactose oxidase C383S mutant identified by directed evolution

Summary for 1T2X

• Entry DOI: 10.2210/pdb1t2x/pdb
• Related: 1GOG 1K3I
• Descriptor: Galactose Oxidase, SODIUM ION, COPPER (II) ION, ... (5 entities in total)
• Functional Keywords: 7 blade beta propeller, c383s mutant form, mutant form of copper containing enzyme, oxidoreductase
• Biological source: Fusarium sp.
• Total number of polymer chains: 1
• Total formula weight: 68826.20

Authors

Wilkinson, D.,Akumanyi, N.,Hurtado-Guerrero, R.,Dawkes, H.,Knowles, P.F.,Phillips, S.E.V.,McPherson, M.J. (deposition date: 2004-04-23, release date: 2004-05-18, Last modification date: 2024-10-09)

Primary citation

Wilkinson, D.,Akumanyi, N.,Hurtado-Guerrero, R.,Dawkes, H.,Knowles, P.F.,Phillips, S.E.V.,McPherson, M.J.
Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.
Protein Eng.Des.Sel., 17:141-148, 2004

PubMed Abstract: 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.

PubMed: 15047910
DOI: 10.1093/protein/gzh018

Experimental method

X-RAY DIFFRACTION (2.3 Å)