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J. Biol. Chem., Vol. 278, Issue 32, 29515-29524, August 8, 2003

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MiaB Protein from Thermotoga maritima

CHARACTERIZATION OF AN EXTREMELY THERMOPHILIC tRNA-METHYLTHIOTRANSFERASE^*

Fabien Pierrel , Heather L. Hernandez , Michael K. Johnson , Marc Fontecave  ¶ and Mohamed Atta  ||

From the Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Département Réponse et Dynamique Cellulaires-Chimie Biologique (DRDC-CB), UMR 5047 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, Commissariat à l'Energie Atomique/Grenoble 17 avenue des Martyrs, 38054 Grenoble Cedex 09, France, and the Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602

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.

Received for publication, February 12, 2003 , and in revised form, May 21, 2003.

^* This work was supported by National Institutes of Health Grant GM62542 (to M. K. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed: DRDC-CB, Commissariat à l'Energie Atomique-Grenoble, 38054 Grenoble, France. Fax: 33-438789124; E-mail: mfontecave{at}cea.fr.

^|| To whom correspondence may be addressed: DRDC-CB, Commissariat à l'Energie Atomique-Grenoble, 38054 Grenoble, France. Fax: 33-438789124; E-mail: mohamed.atta{at}cea.fr.

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