Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase

Bradley J. Landgraf, Squire J. Booker

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 (-SCH 3 ) 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.

Original languageEnglish (US)
Pages (from-to)2889-2892
Number of pages4
JournalJournal of the American Chemical Society
Volume138
Issue number9
DOIs
StatePublished - Mar 9 2016

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S-Adenosylmethionine
Deuterium
Proteins
Bacterial Small Ribosome Subunits
Atoms
Bearings (structural)
Flavodoxin
Oxidants
Aspartic Acid
Isotopes
Escherichia coli
Peptides
Hydrogen
Enzymes
Electrons
Kinetics
Bacteroides thetaiotaomicron

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

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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 (-SCH 3 ) 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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Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase. / Landgraf, Bradley J.; Booker, Squire J.

In: Journal of the American Chemical Society, Vol. 138, No. 9, 09.03.2016, p. 2889-2892.

Research output: Contribution to journalArticle

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