Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei

Abstract

A subset of methanogenic archaea synthesize the cysteinyl-tRNA Cys (Cys-tRNACys) 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-tRNACys (Sep-tRNACys) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNACys 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 tRNACys isoacceptors, further indicating the unusual and complex nature of Cys-tRNACys synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNACys 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 tRNACys species in M. mazei play important roles in the cellular physiology of the organism. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.