Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB

Abstract

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) 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 ms²i⁶A modification is installed onto isopentenyladenosine (i⁶A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe₄S₄]_RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C² hydrogen of the substrate⁵. MiaB also contains an auxiliary [Fe₄S₄]_aux cluster, which has been implicated^6-9 in sulfur transfer to C² of i⁶A37. 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 5'-deoxyadenosyl 5'-radical, which abstracts the C² hydrogen of the substrate but only after C² has undergone rehybridization from sp² to sp³. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.