Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme.
Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH(2) binding. The k(cat)/K(m) value for the C54S SsuD variant increased 3-fold, whereas the k(cat)/K(m) value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH(2) was mixed against C54S SsuD (k(obs), 111 s(-1)) in oxygenated buffer that was 10-fold faster than wild-type SsuD (k(obs), 12.9 s(-1)). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.
Carpenter
Russell A
RA
The Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
Zhan
Xuanzhi
X
Ellis
Holly R
HR
D000476:Alkanesulfonates / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism
D000476:Alkanesulfonates; D029968:Escherichia coli Proteins; D005415:Flavins; D006899:Mixed Function Oxygenases; C499579:SsuD protein, E coli; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine