2VZ3

bleached galactose oxidase

Summary for 2VZ3

• Entry DOI: 10.2210/pdb2vz3/pdb
• Related: 1GOF 1GOG 1GOH 1K3I 1T2X 2VZ1
• Descriptor: GALACTOSE OXIDASE, ACETATE ION, COPPER (II) ION, ... (4 entities in total)
• Functional Keywords: oxidoreductase, copper, secreted, kelch repeat, metal-binding, galactose oxidase, anaerobic processing, thioether bond, thio-ether bond
• Biological source: GIBBERELLA ZEAE
• Cellular location: Secreted: Q01745
• Total number of polymer chains: 1
• Total formula weight: 68887.32

Authors

Rogers, M.S.,Hurtado-Guerrero, R.,Firbank, S.J.,Halcrow, M.A.,Dooley, D.M.,Phillips, S.E.V.,Knowles, P.F.,McPherson, M.J. (deposition date: 2008-07-29, release date: 2008-09-16, Last modification date: 2011-07-13)

Primary citation

Rogers, M.S.,Hurtado-Guerrero, R.,Firbank, S.J.,Halcrow, M.A.,Dooley, D.M.,Phillips, S.E.V.,Knowles, P.F.,McPherson, M.J.
Cross-Link Formation of the Cysteine 228-Tyrosine 272 Catalytic Cofactor of Galactose Oxidase Does not Require Dioxygen.
Biochemistry, 47:10428-, 2008

PubMed 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.

PubMed: 18771294
DOI: 10.1021/BI8010835

Experimental method

X-RAY DIFFRACTION (1.9 Å)