Description:
<jats:p>MiaB catalyzes the transfer of a methylthio (‐SCH<jats:sub>3</jats:sub>) group to C2 of <jats:italic>N<jats:sup>6</jats:sup></jats:italic>‐(isopentenyl)adenosine (i<jats:sup>6</jats:sup>A) in the anticodon stem loop of many prokaryotic tRNAs. The product of the reaction, C2‐methylthio‐<jats:italic>N<jats:sup>6</jats:sup></jats:italic>‐(isopentenyl)adenosine (ms<jats:sup>2</jats:sup>i<jats:sup>6</jats:sup>A) tRNA, is involved in reading frame maintenance and translational fidelity. MiaB is a member of the methylthiotransferase (MTTase) subclass of the radical <jats:italic>S</jats:italic>‐adenosyl‐L‐methionine (SAM) super‐family of enzymes, and as such, contains a canonical [4Fe‐4S] cluster involved in the reductive cleavage of SAM to generate a 5′‐deoxyadenosyl‐5′‐radical (5′dA) required for catalysis. In addition, the enzyme harbors an auxiliary N‐terminal [4Fe‐4S] that is thought to be the source of the sulfur atom inserted at C2, with the methyl group originating from a second molecule of SAM. Although it was anticipated that the MiaB mechanism involved the initial insertion of a sulfur atom prior to the methylation step by SAM to form ms<jats:sup>2</jats:sup>i<jats:sup>6</jats:sup>A, our work indicates that the sequence of mechanistic steps occurs in reversed order. Evidence for the initial methyl‐transfer from SAM to an acid‐labile sulfur atom acceptor present in MiaB was obtained under conditions that preclude the formation of the 5′dA radical species and sulfur insertion. The kinetic competence of the methylated intermediate was assessed by LC/MS of ms<jats:sup>2</jats:sup>i<jats:sup>6</jats:sup>A formation in assays using SAM during the initial methylation of MiaB, and <jats:italic>S</jats:italic>‐adenosyl‐L‐[<jats:italic>methyl‐d<jats:sub>3</jats:sub></jats:italic>]methionine (<jats:italic>d<jats:sub>3</jats:sub></jats:italic>‐SAM) under turnover conditions. Utilizing a combination of radioisotope‐labeling studies, mass spectrometry and spectroscopic techniques, insights into the MiaB mechanism presented here illustrates a new strategy in the diverse repertoire employed by radical SAM enzymes.</jats:p>