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

Abstract

The last biosynthetic step for 2-methylthio-N6-isopen∥ tenyl-adenosine (ms2i6A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopente∥ nyl-adenosine (i6A) precursor. In this work we have re∥ constituted in vitro the conversion of i6A to ms2i6A within a tRNA substrate using the iron-sulfur MiaB pro∥ tein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i6A- containing RNA cor∥ responding to the anticodon stem loop of tRNA Phc is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i 6A. 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 "Rad∥ ical-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.