title: FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase.
abstract: StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.
authors.affiliation: Interdisciplinary Ecological Center, TU Bergakadmie Freiberg, Leipziger Str. 29, 09599 Freiberg, Germany; Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands; Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, United States. Electronic address: dirk-tischler@email.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tischler
authors.forename: Michael
authors.initials: M
authors.lastname: Schlömann
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: George T
authors.initials: GT
authors.lastname: Gassner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012240:Rhodococcus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C023111:epoxidase; D010105:Oxygenases; C026721:styrene monooxygenase
keywords: Catalytic mechanism; FAD (C4a)-hydroperoxide and FAD C(4a)-hydroxide; FAD(ox) and FAD(red); FADH(OOH) and FADH(OH); Flavoprotein; Monooxygenase; Rhodococcus opacus 1CP; SMO; StyA and StyB; StyA2B; Styrene epoxidation; epoxidase and reductase subunit of conventional two-component SMOs; naturally fused self-sufficient SMO comprising reductase (B) and epoxidase (A2); oxidized and reduced FAD; styrene monooxygenase