†Department
of Chemistry, ‡Department of Biochemistry and Molecular Biology, and §The Howard Hughes
Medical Institute, The Pennsylvania State
University, University Park, Pennsylvania 16802, United States
J. Am. Chem. Soc., Article ASAP
DOI: 10.1021/jacs.5b11035
Abstract
RimO
is a member of the growing radical S-adenosylmethionine (SAM)
superfamily of enzymes, which use a reduced [4Fe–4S] cluster to effect
reductive cleavage of the 5′ C–S bond of SAM to form a 5′-deoxyadenosyl
5′-radical (5′-dA•) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (−SCH3)
to C3 of aspartate 89 of protein S12, one of 21 proteins that compose
the 30S subunit of the bacterial ribosome. However, the exact mechanism
by which this transformation takes place has remained elusive. Herein,
we describe the stereochemical course of the RimO reaction. Using
peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S
hydrogen atom, as evidenced by the transfer of deuterium into
5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H
atom versus D atom abstraction is ∼1.9, suggesting that this step is at
least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product.
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