Biocatalytic properties and structural analysis of eugenol oxidase from Rhodococcus jostii RHA1: a versatile oxidative biocatalyst

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 was previously shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and hydroxylation of eugenol, EUGO can efficiently perform the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol, 4-(hydroxy-1-ethyl)-2-methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complex with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent-inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl-alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p-substituents while allowing o-methoxy substituents.