Structure-Based Enzyme Tailoring of 5-Hydroxymethylfurfural Oxidase

5-Hydroxymethylfurfural oxidase (HMFO) is a flavin-dependent enzyme that catalyzes the oxidation of many aldehydes, primary alcohols, and thiols. The three-step conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid is relevant for the industrial production of biobased polymers. The remarkable wide substrate scope of HMFO contrasts with the enzyme’s precision in positioning the substrate to perform catalysis. We have solved the crystal structure of HMFO at 1.6 Å resolution, which guided mutagenesis experiments to probe the role of the active-site residues in catalysis. Mutations targeting two active-site residues generated engineered forms of HMFO with promising catalytic features, namely enantioselective activities on secondary alcohols and improved 2,5-furandicarboxylic acid yields.