Abstract
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 (m1)G37 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.
Highlights
Some marine environments, the Methanosarcina flourish together with methanotrophs and sulfate-reducing bacteria, where they participate in a microbial community that generates very high sulfate reduction rates via anaerobic methane oxidation [3]
Other work on the M. jannaschii pathway has shown that synthesized by phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS) form a stable binary complex that assists in conversion of the intermediate SeptRNACys to Cys-tRNACys, a complex reminiscent of the transamidosome particle that facilitates prokaryotic tRNA-dependent asparagine biosynthesis [11, 12]
By examining aminoacylation of the three wild-type as well as a set of hybrid tRNACys species as substrates for either cysteinyl-tRNA synthetase (CysRS) or the combined SepRS/SepCysS pathway, we show that each enzyme prefers a distinct tRNACys isoacceptor or pair of isoacceptors and that the nucleotides determining CysRS versus SepRS selectivity among tRNACys species are located in the globular core of the tRNA
Summary
Some marine environments, the Methanosarcina flourish together with methanotrophs and sulfate-reducing bacteria, where they participate in a microbial community that generates very high sulfate reduction rates via anaerobic methane oxidation [3]. TRNACys is first aminoacylated with the nonstandard amino acid phosphoserine by phosphoseryl-tRNA synthetase (SepRS).2 The misacylated Sep-tRNACys is converted to Cys-tRNACys through the action of the PLP-dependent enzyme SepCysS.
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