title: Identification of an intermediate methyl carrier in the radical S-adenosylmethionine methylthiotransferases RimO and MiaB.
abstract: RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (-SCH3) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30S subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of N(6)-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM enzymes called methylthiotransferases (MTTases). It had been assumed that the sequence of steps in MTTase reactions involves initial sulfur insertion into the organic substrate followed by capping of the inserted sulfur atom with a SAM-derived methyl group. In this work, however, we show that both RimO and MiaB from Thermotoga maritima catalyze methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of their respective macromolecular substrates. Consistent with the assignment of the acceptor as an iron-sulfur cluster, denaturation of the SAM-treated protein with acid results in production of methanethiol. When RimO or MiaB is first incubated with SAM in the absence of substrate and reductant and then incubated with excess S-adenosyl-l-[methyl-d3]methionine in the presence of substrate and reductant, production of the unlabeled product precedes production of the deuterated product, showing that the methylated species is chemically and kinetically competent to be an intermediate.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Landgraf
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Arcinas
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States.
authors.forename: Kyung-Hoon
authors.initials: KH
authors.lastname: Lee
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802, United States.
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D015394:Molecular Structure; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D013438:Sulfhydryl Compounds; C005231:methylmercaptan; D012436:S-Adenosylmethionine