Engineering of galactose oxidase substrate specificity (360G-Wellcome-089309_Z_09_Z)

Amadori-modification of serum proteins occurs at much higher levels in diabetic patients than in normal individuals and the resulting glycation can lead to a number of complications such as atherosclerosis and cataract formation (1). Accurate monitoring of serum protein glycation may enable diagnosis of diabetes in its early stages. Galactose oxidase (GO) from the fungus Fusarium graminearum has been extensively characterised and shown to have a solvent accessible active site and broad substrate specificity making it an ideal candidate for the introduction of activity with new substrates. Following further characterisation of the enzyme to broaden our understanding of key residues, we aim to engineer GO to accept glycated serum albumin as a substrate in order to enable future development of a biosensor to be used in the diagnosis of diabetes. We also aim to introduce activity against other sugar substrates with potential uses in chemical synthesis and pharmaceutical industries. Objective 1) Develop an agar plate-based assay to screen colonies containing mutants of GO for oxidase activity. Objective 2) Determine crystallisation conditions for E. coli-expressed GO. Objective 3) Explore the role of selected second sphere residues in the catalytic function and substrate specificity of GO. Objective 4) Through modification of active site loop regions, engineer the enzyme to accept alternative substrates, including Amadori-modified serum proteins which are elevated in diabetic patients.