BIOL 176 |
| The two-component alkanesulfonate monooxygenase system from Escherichia coli belongs to a family of enzymes that utilize FMN as a substrate rather than a bound prosthetic group. This two-component system that includes an FMN reductase (SsuE) and an reduced FMN-dependent alkanesulfonate monooxygenase (SsuD) is involved in the conversion of alkanesulfonate to sulfite and the corresponding aldehyde during times of sulfur limitation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of reduced FMN and O2. Although the mechanism of SsuE has been characterized, the mechanism of the monooxygenase enzyme in this system is not well understood. The goal of these studies was to investigate the kinetic mechanism of SsuD through substrate binding studies and rapid reaction kinetics. Reduced flavin mononucleotide (reduced FMN) is the first substrate that binds to SsuD with a ratio of 1:1. Results from fluorimetric titrations imply that octanesulfonate is not able to bind to SsuD enzyme unless reduced FMN is first bound. In stopped-flow diode array and single-wavelength analysis, there is no direct evidence that a 4a-hydroperyxyflavin intermediate is generated in the desulfonation reaction. This intermediate has been identified as the reactive oxygen species in other monooxygenases from flavin-dependent two-component systems. Results from rapid reaction kinetic studies show that an induced conformational change is likely involved in the binding of FMNH2 and octanesulfonate. These results suggest an ordered substrate binding mechanism to SsuD, and this substrate binding order plays an essential catalytic role in the desulfonation reaction. |
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Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |