Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase.
Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1). Single turnover studies of the reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance characteristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find that the observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer and passive diffusion of reduced FAD from SMOB to SMOA.
Kantz
Auric
A
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132-4163, USA.
Chin
Franklin
F
Nallamothu
Nagamani
N
Nguyen
Tim
T
Gassner
George T
GT
D001554:Benzene / Q000037:antagonists & inhibitors; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D016958:Pseudomonas putida / Q000378:metabolism*; D020058:Styrene / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005415:Flavins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D020058:Styrene; D006639:Histidine; D010088:Oxidoreductases; C023111:epoxidase; D010105:Oxygenases; C026721:styrene monooxygenase; D001554:Benzene; D010100:Oxygen