PDBsum entry 2vz3

Oxidoreductase

PDB id: 2vz3

Contents

Protein chain

639 a.a. *

Ligands

ACT ×2

Metals

_CU ×3

Waters ×390

* Residue conservation analysis

PDB id:

2vz3

Name:

Oxidoreductase

Title:

Bleached galactose oxidase

Structure:

Galactose oxidase. Chain: a. Synonym: gao, gaoase, go, bleached galactose oxidase. Engineered: yes

Source:

Gibberella zeae. Organism_taxid: 5518. Expressed in: pichia pastoris. Expression_system_taxid: 4922.

Resolution:

1.90Å R-factor: 0.169 R-free: 0.203

Authors:

M.S.Rogers,R.Hurtado-Guerrero,S.J.Firbank,M.A.Halcrow,D.M.Dooley, S.E.V.Phillips,P.F.Knowles,M.J.Mcpherson

Key ref:

M.S.Rogers et al. (2008). Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen. Biochemistry, 47, 10428-10439. PubMed id: 18771294

Date:

29-Jul-08 Release date: 16-Sep-08

Protein chain

P0CS93  (GAOA_GIBZA) -  Galactose oxidase from Gibberella zeae

Seq: 680 a.a.

Struc: 639 a.a.

Enzyme reactions

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

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

reference

Biochemistry 47:10428-10439 (2008)

PubMed id: 18771294

Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen.

M.S.Rogers, R.Hurtado-Guerrero, S.J.Firbank, M.A.Halcrow, D.M.Dooley, S.E.Phillips, P.F.Knowles, M.J.McPherson.

ABSTRACT

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda max = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.