title: Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis.
abstract: Catalysis for chemical synthesis by cell-free monooxygenases necessitates an efficient and robust in situ regeneration system to supply the enzyme with reducing equivalents. We report on a novel approach to directly regenerate flavin-dependent monooxygenases. The organometallic complex [CpRh(bpy)(H(2)O)](2+) catalyzes the transhydrogenation reaction between formate and isoalloxazine-based cofactors such as FAD and FMN. Coupling this FADH(2) regeneration reaction to the FADH(2)-dependent styrene monooxygenase (StyA) resulted in a chemoenzymatic epoxidation reaction where the organometallic compound substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an artificial NADH regeneration system such as formate dehydrogenase. Various styrene derivatives were converted into the essentially optically pure (S)-epoxides (ee > 98%). In addition, StyA was shown to be capable of performing sulfoxidation reactions. The productivity of the chemoenzymatic epoxidation reaction using 6.5 microM StyA reached up to 6.4 mM/h, corresponding to approximately 70% of a comparable fully enzymatic reaction using StyB, NADH, and formate dehydrogenase for regeneration. The coupling efficiency of the nonenzymatic regeneration reaction to enzymatic epoxidation was examined in detail, leading to an optimized reaction setup with minimized quenching of the electron supply for the epoxidation reaction. Thus, up to 60% of the reducing equivalents provided via [CpRh(bpy)(H(2)O)](2+) could be channeled into epoxide rather than hydrogen peroxide formation, allowing selective synthesis with high yields.
authors.affiliation: Institute of Biotechnology, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland.
authors.forename: Frank
authors.initials: F
authors.lastname: Hollmann
authors.forename: Po-Chi
authors.initials: PC
authors.lastname: Lin
authors.forename: Bernard
authors.initials: B
authors.lastname: Witholt
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
mesh_terms: D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004852:Epoxy Compounds / Q000138:chemical synthesis*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism; D012238:Rhodium / Q000737:chemistry; D013343:Styrenes / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: C476958:(2,2'-bipyridyl)(pentamethylcyclopentadienyl)rhodium; D004852:Epoxy Compounds; D009942:Organometallic Compounds; D013343:Styrenes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D012238:Rhodium; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C026721:styrene monooxygenase