Identification of an Intermediate Methyl Carrier and the Stereochemical Outcomes of H‐atom Abstraction and Methylthiolation by the Radical SAM Enzyme RimO
You can manage bookmarks using lists, please log in to your user account for this.
Media type:
E-Article
Title:
Identification of an Intermediate Methyl Carrier and the Stereochemical Outcomes of H‐atom Abstraction and Methylthiolation by the Radical SAM Enzyme RimO
Description:
<jats:p>RimO is a radical <jats:italic>S</jats:italic>‐adenosylmethionine (RS) enzyme that catalyzes the insertion of a methylthio group (‐SCH<jats:sub>3</jats:sub>) at C3 of a conserved aspartyl residue of protein S12<jats:italic>,</jats:italic> a component of the 30<jats:italic>S</jats:italic> subunit of the bacterial ribosome<jats:italic>.</jats:italic> RimO contains a [4Fe‐4S] cluster to which <jats:italic>S</jats:italic>‐adenosylmethionine (SAM) binds, allowing for its reductive cleavage to methionine and a 5′‐deoxyadenosyl 5′‐radical. Unlike most RS enzymes, methylthiotransferases (MTTases) contain an additional [4Fe–4S] cluster that is believed to be the source of the sulfur atom of the inserted –SCH<jats:sub>3</jats:sub> group. It was believed that the sequence of MTTase reactions involved initial sulfur insertion into the substrate followed by capping with a SAM‐derived methyl group. We show that RimO from <jats:italic>Thermotoga maritima</jats:italic> catalyzes the synthesis and subsequent insertion of a ‐SCH<jats:sub>3</jats:sub> group by mediating methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of its substrate and the requisite reductant that triggers radical chemistry. Consistent with the assignment of the acceptor as an Fe‐S cluster (presumably the auxiliary [4Fe–4S] cluster), denaturation of the SAM‐treated protein with acid results in production of methanethiol (CH<jats:sub>3</jats:sub>SH). We show chemical competence of the methylated intermediate by using unlabeled and [<jats:italic>methyl</jats:italic>‐<jats:italic>d<jats:sub>3</jats:sub>]</jats:italic> SAM and demonstrate that RimO catalyzes multiple turnovers when CH<jats:sub>3</jats:sub>SH is supplied exogenously. Using deuterated isotopologs at C3 of aspartate and <jats:sup>1</jats:sup>H NOESY NMR, we determined the stereospecificity of H‐atom abstraction and the absolute configuration of the chiral center created upon ‐SCH<jats:sub>3</jats:sub> insertion by RimO.</jats:p>