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 isopentenyl-adenosine (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 tRNA(Phe) 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 ofa5'-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.
Pierrel
Fabien
F
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DRDC-CB, Unité Mixte de Recherche 5047 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, Grenoble, France.
Douki
Thierry
T
Fontecave
Marc
M
Atta
Mohamed
M
D000241:Adenosine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000378:metabolism
D029968:Escherichia coli Proteins; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; C499584:MiaB protein, E coli; D013466:Sulfurtransferases; C402430:isopentenyl-adenosine i6a thiotransferase; D000241:Adenosine