The flavin mononucleotide cofactor in α -hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α -ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C ′ α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α -hydroxyamide or α -ketoamide biochemically and structurally supports the proposed mechanism.

Source: Acta Crystallographica Section D - September 23, 2019 Category: Biochemistry Authors: Lyu, S.-Y. Lin, K.-H. Yeh, H.-W. Li, Y.-S. Huang, C.-M. Wang, Y.-L. Shih, H.-W. Hsu, N.-S. Wu, C.-J. Li, T.-L. Tags: electrophilic/nucleophilic duality α -hydroxyacid oxidases flavin mononucleotide oxidative decarboxylation monooxygenase p-hydroxymandelate oxidase research papers Source Type: research