Publications

• Structure and mechanism of galactose oxidase. The free radical site.

  Baron A.J., Stevens C., Wilmot C., Seneviratne K.D., Blakeley V., Dooley D.M., Phillips S.E., Knowles P.F., McPherson M.J.

  Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, ... >> More

  Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site. << Less

  J. Biol. Chem. 269:25095-25105(1994) [PubMed] [EuropePMC]

• The D-galactose oxidase of Polyporus circinatus.

  Avigad G., Amaral D., Asensio C., Horecker B.L.

  J. Biol. Chem. 237:2736-2743(1962) [PubMed] [EuropePMC]

• Enhanced fructose oxidase activity in a galactose oxidase variant.

  Deacon S.E., Mahmoud K., Spooner R.K., Firbank S.J., Knowles P.F., Phillips S.E., McPherson M.J.

  Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previousl ... >> More

  Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO. << Less

  ChemBioChem 5:972-979(2004) [PubMed] [EuropePMC]

• The stacking tryptophan of galactose oxidase: a second-coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis.

  Rogers M.S., Tyler E.M., Akyumani N., Kurtis C.R., Spooner R.K., Deacon S.E., Tamber S., Firbank S.J., Mahmoud K., Knowles P.F., Phillips S.E.V., McPherson M.J., Dooley D.M.

  The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is sign ... >> More

  The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower in the W290 variants. The Km for D-galactose for W290H is similar to that of the wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in the W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes are also closely similar to the wild-type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function. << Less

  Biochemistry 46:4606-4618(2007) [PubMed] [EuropePMC]

• 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 P.F., Phillips S.E.V., McPherson M.J.

  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 express ... >> More

  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. << Less

  Protein Eng. Des. Sel. 17:141-148(2004) [PubMed] [EuropePMC]