MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA

The last biosynthetic step for 2-methylthio-N-6-isopentenyl-adenosine (ms(2)i(6)A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyladenosine (i(6)A) precursor. In this work we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i(6)A-containing RNA corresponding to the anticodon stem loop of tRNAPhe is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i(6)A. In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation of a 5'-deoxyadenosyl radical as observed in other Radical-AdoMet enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.