title: Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase.
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.
authors.affiliation: 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.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Landgraf
authors.affiliation: 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.
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013466:Sulfurtransferases / Q000737:chemistry* / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D029968:Escherichia coli Proteins; D005418:Flavodoxin; D012436:S-Adenosylmethionine; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase; C545011:RimO protein, E coli; D013466:Sulfurtransferases