MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase.
In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37). For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates. The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E. coli. MiaBTm was purified to homogeneity and thoroughly characterized by biochemical and spectroscopic methods. It is a monomer of 443 residues with a molecular mass of 50,710 kilodaltons. Its amino acid sequence shares the CysXXX-CysXXCys sequence with MiaB from E. coli as well as with biotin synthase and lipoate synthase. This sequence was shown to be essential for chelation of an iron-sulfur center and for activity in these enzymes. As isolated, MiaBTm contains both iron and sulfide and an apoprotein form can coordinate up to 4 iron and 4 sulfur atoms per polypeptide chain. UV-visible absorption, resonance Raman, variable temperature magnetic circular dichroism, and EPR spectroscopy of MiaBTm indicate the presence of a [4Fe-4S]+2/+1 cluster under reducing and anaerobic conditions, whereas [3Fe-4S]+1 and [2Fe-2S]+2 forms are generated under aerobic conditions. The redox potential of the [4Fe-4S]+2/+1 transition is -495 +/- 10 mV (versus the normal hydrogen electrode). Finally, the expression of MiaBTm from T. maritima in an E. coli mutant strain lacking functional miaB gene allowed production of ms2i6A-37. These results provide further information on the enzymes involved in methylthiolation of tRNAs.
Pierrel
Fabien
F
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Département Réponse et Dynamique Cellulaires-Chimie Biologique, UMR 5047 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, Commissariat à l'Energie Atomique/Grenoble, France.
Hernandez
Heather L
HL
Johnson
Michael K
MK
Fontecave
Marc
M
Atta
Mohamed
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010453:Peptide Synthases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013466:Sulfurtransferases / Q000737:chemistry*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*; D013997:Time Factors; D014466:Ultraviolet Rays; D012359:tRNA Methyltransferases / Q000737:chemistry*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C499584:MiaB protein, E coli; D013466:Sulfurtransferases; C402430:isopentenyl-adenosine i6a thiotransferase; C057985:biotin synthetase; D010453:Peptide Synthases; C072322:lipoate-protein ligase; D003545:Cysteine