PDBsum entry: 1t2x

Oxidoreductase

PDB id: 1t2x

Contents

Protein chain

639 a.a. *

Ligands

ACT ×3

Metals

_NA

_CU

Waters ×313

* Residue conservation analysis

PDB id:

1t2x

Name:

Oxidoreductase

Title:

Glactose oxidase c383s mutant identified by directed evolution

Structure:

Galactose oxidase. Chain: a. Engineered: yes. Mutation: yes

Source:

Fusarium sp.. Organism_taxid: 29916. Gene: sac1. Expressed in: pichia pastoris. Expression_system_taxid: 4922.

Resolution:

2.30Å R-factor: 0.218 R-free: 0.228

Authors:

D.Wilkinson,N.Akumanyi,R.Hurtado-Guerrero,H.Dawkes, P.F.Knowles,S.E.V.Phillips,M.J.Mcpherson

Key ref:

D.Wilkinson et al. (2004). Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution. Protein Eng Des Sel, 17, 141-148. PubMed id: 15047910 DOI: 10.1093/protein/gzh018

Date:

23-Apr-04 Release date: 18-May-04

Protein chain

Q01745  (GAOA_GIBZE) -  Galactose oxidase

Seq: 680 a.a.

Struc: 639 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.1.3.9 - Galactose oxidase.
• Reaction: D-galactose + O₂ = D-galacto-hexodialdose + H₂O₂
• Cofactor: Copper

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biological process: cell adhesion (2 terms)
• Biochemical function: oxidoreductase activity (3 terms)

reference

DOI no: 10.1093/protein/gzh018

Protein Eng Des Sel 17:141-148 (2004)

PubMed id: 15047910

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

D.Wilkinson, N.Akumanyi, R.Hurtado-Guerrero, H.Dawkes, P.F.Knowles, S.E.Phillips, M.J.McPherson.

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.