Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase.
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 CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.
Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT, CH-8093, Zurich, Switzerland.
Katja
K
Otto
Karin
K
Hofstetter
Martina
M
Röthlisberger
Bernard
B
Witholt
Andreas
A
Schmid
D003001:Cloning, Molecular; D019281:Dimerization; D004579:Electron Transport; D004852:Epoxy Compounds / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase / Q000302:isolation & purification* / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D020058:Styrene / Q000378:metabolism*; D013379:Substrate Specificity
D004852:Epoxy Compounds; D021122:Protein Subunits; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D020058:Styrene; D005486:Flavin Mononucleotide; C013690:styrene oxide; D010105:Oxygenases; C026721:styrene monooxygenase; D038181:FMN Reductase; D012256:Riboflavin