Catalytic importance of the substrate binding order for the FMNH2-dependent alkanesulfonate monooxygenase enzyme.
The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 (Kd, 0.32 +/- 0.15 microM) compared to FMN (Kd, 10.2 +/- 0.4 microM) with a 1:1 binding stoichiometry for each form of the flavin. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. When octanesulfonate was included in the kinetic studies the C4a-(hydro)peroxyflavin was observed at 370 nm when FMNH2 was not premixed with SsuD, which correlated with an increase in octanal product. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate.
Zhan
Xuanzhi
X
Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
Carpenter
Russell A
RA
Ellis
Holly R
HR
D002384:Catalysis; D029968:Escherichia coli Proteins / Q000378:metabolism*; D038181:FMN Reductase / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D005415:Flavins; D006899:Mixed Function Oxygenases; C499579:SsuD protein, E coli; D038181:FMN Reductase; C447459:SsuE protein, E coli; D009247:NADH, NADPH Oxidoreductases