bi300138d_si_001.pdf (93.34 kB)
Identification of Critical Steps Governing the Two-Component Alkanesulfonate Monooxygenase Catalytic Mechanism
journal contribution
posted on 2012-08-14, 00:00 authored by John M. Robbins, Holly R. EllisThe alkanesulfonate monooxygenase enzyme (SsuD) catalyzes
the oxygenolytic
cleavage of a carbon–sulfur bond from sulfonated substrates.
A mechanism involving acid–base catalysis has been proposed
for the desulfonation mechanism by SsuD. In the proposed mechanism,
base catalysis is involved in abstracting a proton from the alkane
peroxyflavin intermediate, while acid catalysis is needed for the
protonation of the FMNO– intermediate. The pH profiles
of kcat indicate that catalysis by SsuD
requires a group with a pKa of 6.6 ±
0.2 to be deprotonated and a second group with a pKa of 9.5 ± 0.1 to be protonated. The upper pKa value was not present in the pH profiles of kcat/Km. Several
conserved amino acid residues (His228, His11, His333, Cys54, and Arg226)
have been identified as having potential catalytic importance due
to the similar spatial arrangements with close structural and functional
relatives of SsuD. Substitutions to these amino acid residues were
generated, and the pH dependencies were evaluated and compared to
wild-type SsuD. Although a histidine residue was previously proposed
to be the active site base, the His variants possessed similar steady-state
kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K
SsuD variants possessed undetectable activity, and there was no detectable
formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226
SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted
in the recovery of 1.5% of the wild-type SsuD kcat value. These results implicate Arg226 playing a critical
role in catalysis and provide essential insights into the mechanistic
steps that guide the SsuD desulfonation process.