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Reaction mechanism studies of alkanesulfonate monooxygenase from Escherichia coli
Author(s) -
Zhan Xuanzhi,
Ellis Holly R.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb51-b
Subject(s) - chemistry , monooxygenase , flavin group , sulfite , substrate (aquarium) , aldehyde , steady state (chemistry) , reaction rate constant , enzyme kinetics , stereochemistry , kinetics , catalysis , half reaction , flavin mononucleotide , reaction mechanism , enzyme , reactivity (psychology) , active site , organic chemistry , cytochrome p450 , biology , physics , quantum mechanics , medicine , ecology , alternative medicine , pathology
The two‐component alkanesulfonate monooxygenase system from Escherichia coli belongs to a family of enzymes that utilizes FMN as a substrate rather than a bound prosthetic group. This two‐component system that includes an FMN reductase (SsuE) and an FMNH 2 ‐dependent alkanesulfonate moonoxygenase (SsuD) are involved in the conversion of alkanesulfonate to sulfite and the corresponding aldehyde. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of FMNH 2 and O 2 . The primary objective of this study is to elucidate the kinetic mechanism of this enzymatic reaction through rapid reaction kinetics. In the absence of alkanesulfonates, a 4a‐(hydro)peroxyflavin intermediate has been identified by the reaction of premixed SsuD and reduced flavin with molecular oxygen. The formation of 4a‐(hydro)peroxyflavin intermediate is oxygen‐dependent with a second order rate constant of 2.35×10 4 M −1 s −1 . In the absence of alkanesulfonates, this complex decays to FMN and H 2 O 2 at a second order rate of 1.67×10 4 M −1 s −1 . The pre‐steady state results provide evidence for the involvement of a 4a‐(hydro)peroxyflavin intermediate in the SsuD catalytic reaction. Current studies are focused on determining the FMNH 2 binding affinity of SsuD and SsuE, and pre‐steady state analysis of SsuD in the presence of alkanesulfonates. This research is supported by Auburn University.