Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio- N 6 -isopentenyladenosine (ms 2 i 6 A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon–anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity 1 – 4 . The ms 2 i 6 A modification is installed onto isopentenyladenosine (i 6 A) by MiaB, a radical S -adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe 4 S 4 ] RS cluster used in the reductive cleavage of SAM to form a 5ʹ-deoxyadenosyl 5ʹ-radical, which is responsible for removing the C 2 hydrogen of the substrate 5 . MiaB also contains an auxiliary [Fe 4 S 4 ] aux cluster, which has been implicated 6 – 9 in sulfur transfer to C 2 of i 6 A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis . These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5ʹ-deoxyadenosyl 5ʹ-radical, which abstracts the C 2 hydrogen of the substrate but only after C 2 has undergone rehybridization from sp 2 to sp 3 . This work advances our understanding of how enzymes functionalize inert C–H bonds with sulfur. Crystal structures reveal the catalytic mechanism through which the radical S -adenosylmethionine enzyme MiaB adds a methylthio group onto tRNA.