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Functionalization of Oxidases with Peroxidase Activity Creates Oxiperoxidases: A New Breed of Hybrid Enzyme Capable of Cascade Chemistry
Author(s) -
Winter Remko T.,
van den Berg Tomas E.,
Colpa Dana I.,
van Bloois Edwin,
Fraaije Marco W.
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100639
Subject(s) - peroxidase , chemistry , biochemistry , phosphofructokinase 2 , protein engineering , flavoprotein , directed evolution , enzyme , heme , thermostability , oxidase test , covalent bond , streptomyces coelicolor , combinatorial chemistry , organic chemistry , gene , mutant
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.