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J. Biol. Chem., Vol. 283, Issue 32, 22007-22017, August 8, 2008

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Redundant Synthesis of Cysteinyl-tRNA^Cys in Methanosarcina mazei^*

Scott I. Hauenstein and John J. Perona¹

From the Department of Chemistry and Biochemistry and the Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510

A subset of methanogenic archaea synthesize the cysteinyl-tRNA^Cys (Cys-tRNA^Cys) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA^Cys (Sep-tRNA^Cys) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA^Cys by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA^Cys isoacceptors, further indicating the unusual and complex nature of Cys-tRNA^Cys synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA^Cys isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of ^m1G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA^Cys species in M. mazei play important roles in the cellular physiology of the organism.

Received for publication, March 6, 2008 , and in revised form, May 19, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM63713 (to J. J. P.). 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.

¹ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, CA 93106-9510. Fax: 805-893-4120; E-mail: perona{at}chem.ucsb.edu.

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