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

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 kcat 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

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

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 kcat 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

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

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 kcat 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

Vanadium-Containing Chloroperoxidase-Catalyzed Versatile Valorization of Phenols and Phenolic Acids

The downstream product transformation of lignin depolymerization is of great interest in the production of high-value aromatic chemicals. However, this transformation is often impeded by chemical oxidation under harsh reaction conditions. In this study, we demonstrate that hypohalites generated in situ by the vanadium-containing chloroperoxidase from Curvularia inaequalis (CiVCPO) can halogenate various electron-rich and electron-poor phenol and phenolic acid substrates. Specifically, CiVCPO enabled decarboxylative halogenation, deformylative halogenation, halogenation, and direct oxidation reactions. The versatile transformation routes for the valorization of phenolic compounds showed up to 99% conversion and 99% selectivity, with a turnover number of 60,700 and a turnover frequency of 60 s–1 for CiVCPO. This study potentially expands the biocatalytic toolbox for lignin valorization