1t2x

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Glactose oxidase C383S mutant identified by directed evolution

Structural highlights

1t2x is a 1 chain structure with sequence from Fusarium sp.. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.3Å
Ligands:ACT, CU, NA
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

GAOA_GIBZA Catalyzes the sterospecific oxidation of primary alcohols to the corresponding aldehydes. The biologically relevant substrate of the enzyme is not known as the enzyme exhibits broad substrate specificity from small alcohols through sugars to oligo- and polysaccharides.[1] [2]

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

Publication Abstract from PubMed

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.

Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.,Wilkinson D, Akumanyi N, Hurtado-Guerrero R, Dawkes H, Knowles PF, Phillips SE, McPherson MJ Protein Eng Des Sel. 2004 Feb;17(2):141-8. Epub 2004 Jan 12. PMID:15047910[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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See Also

References

  1. ↑ COOPER JA, SMITH W, BACILA M, MEDINA H. Galactose oxidase from Polyporus circinatus, Fr. J Biol Chem. 1959 Mar;234(3):445-8 PMID:13641238
  2. ↑ Kosman DJ, Ettinger MJ, Weiner RE, Massaro EJ. The molecular properties of the copper enzyme galactose oxidase. Arch Biochem Biophys. 1974 Dec;165(2):456-67. PMID:4441089 doi:10.1016/0003-9861(74)90271-9
  3. ↑ Wilkinson D, Akumanyi N, Hurtado-Guerrero R, Dawkes H, Knowles PF, Phillips SE, McPherson MJ. Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution. Protein Eng Des Sel. 2004 Feb;17(2):141-8. Epub 2004 Jan 12. PMID:15047910 doi:10.1093/protein/gzh018

Contents


PDB ID 1t2x

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OCA

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