Structure-Based Redesign of a Methanol Oxidase into an “Aryl Alcohol Oxidase” for Enzymatic Synthesis of Aromatic Flavor Compounds

Abstract

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H₂O₂ as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and k_cat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s^-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.