Mechanism of flavin reduction in the alkanesulfonate monooxygenase system.
The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s(-1)) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s(-1)) to form a charge-transfer complex of reduced FMNH(2) with NADP(+) (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s(-1)) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-(2)H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.
Gao
Benlian
B
The Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA.
Ellis
Holly R
HR
D000476:Alkanesulfonates / Q000378:metabolism*; D002384:Catalysis; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology
D000476:Alkanesulfonates; D005415:Flavins; D009249:NADP; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases