Functionalization of oxidases with peroxidase activity creates oxiperoxidases: a new breed of hybrid enzyme capable of cascade chemistry

Abstract

The covalent flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2) was endowed with an extra catalytic functionality by fusing it to a microperoxidase. Purification of the construct resulted in the isolation of a synthetic bifunctional enzyme that was both fully covalently flavinylated and heminylated: an oxiperoxidase. Characterization revealed that both oxidase and peroxidase functionalities were active, with the construct functioning as a single-component xylitol biosensor. In an attempt to reduce the size of the oxidase-peroxidase fusion, we replaced portions of the native AldO sequence with the bacterial cytochrome c CXXCH heme-binding motif. By mutating only three residues of the AldO protein we were able to create a functional oxidase-peroxidase hybrid.