Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase

Abstract

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CAC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

“…It reacts either with molecular oxygen to yield hydrogen peroxide and FAD or with FAD to form highly reactive flavin radicals, which can react with molecular oxygen to form hydrogen peroxide (41). In vitro, uncoupling accounted for up to 80% of the NADH consumption and depended on the FAD concentration and the StyA/StyB ratio (30,47). The extent of uncoupling under in vivo conditions is not known so far.…”

“…Uncoupling might contribute more significantly to high coenzyme consumption rates. Styrene monooxygenase is a twocomponent flavoenzyme composed of the NADH-specific flavin reductase StyB and the reduced flavin adenine dinucleotide (FADH 2 )-dependent styrene epoxidase StyA, with freely diffusible FAD(H 2 ) serving as the electron shuttle between the two subunits (47). Instead of activating molecular oxygen for oxygenation reactions, FADH 2 can be reoxidized in two different types of uncoupling reactions.…”

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

“…It is a member of the class E flavoprotein monooxygenases, which is composed of the oxygenase (StyA) and the reductase (StyB) domains. StyA catalyzes the epoxidation of alkenes, and StyB catalyzes the two-electron reduction of FAD (Kantz et al, 2005;Kantz and Gassner, 2011;Otto et al, 2004). The native substrate of SMO is styrene, which can undergo asymmetric epoxidation to form the single enantiomer of (S)-styrene oxide ( Fig.…”

Rational design of styrene monooxygenase mutants with altered substrate preference

“…Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of a flavin adenine dinucleotide (FAD)-specific styrene epoxidase, StyA (the larger oxygenase domain) that catalyzes the epoxidation of C = C double bonds, and an NADH-specific flavin reductase, StyB (the smaller reductase domain) that catalyzes the two-electron reduction of FAD (Otto et al 2004;Kantz et al 2005). It is a highly enantioselective enzyme that catalyzes the formation of (S)-styrene oxide from styrene with an excellent enantiomeric excess of more than 99% ee (Bernasconi et al 2000;Panke et al 2000;Bernasconi et al 2004;Han et al 2006), which provides an alternative to the generally applied Sharpless-Katsuki epoxidation (Sharpless 2002) and Jacobsen epoxidation (Jacobsen et al 1991) for the synthesis of chiral building blocks for pharmaceuticals or agrochemicals (Noyori et al 2003).…”