Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei

Abstract

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-adenosylmethionedependent 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.

Catalytic Mechanism of Sep-tRNA:Cys-tRNA Synthase

“…Although a S 2Ϫ equivalent is likely a sulfur donor for the persulfidation of SepCysS (described above), the low in vitro sulfhydrylation activity of SepCysS with sodium sulfide suggested that bisulfide is unlikely to be the physiological sulfur donor (2,8). Because SepCysS was able to receive sulfur from the IscS persulfide, an unidentified protein-carrying persulfide possibly provides the sulfur for the persulfidation of SepCysS.…”

“…6). This reaction scheme can also explain the in vitro activity with sulfide, which may directly attack the disulfide of SepCysS or the double bond of the dehydroalanyl moiety (8).…”

Ancient translation factor is essential for tRNA-dependent cysteine biosynthesis in methanogenic archaea

“…However, formation of this complex caused both SepRS and SepCysS to dissociate from their oligomeric states into monomers (15), and thus this complex is unlikely to be physiologically active. Furthermore, addition of SepRS to the SepCysS reaction does not improve CystRNA Cys production (16). Here we present a translation factor, SepCysE, essential for tRNA-dependent Cys biosynthesis in methanococci.…”

“…All steps of the Cys-tRNA Cys formation were performed in an anaerobic chamber with an atmosphere of 95% (vol/vol) N 2 and 5% (vol/vol) H 2 . The assay used 1 mM Na 2 S as the sulfur donor and was carried out as described (16) with modification (SI Materials and Methods).…”

Gene expression microarray analysis encompassing metamorphosis and the onset of calcification in the scleractinian coral Montastraea faveolata

“…Other pattern 8 genes that may be involved in OM synthesis and calcification include zinc metalloproteinase nas-13 (CAON980 -an astacin-like metalloproteinase) and cysteinyl-tRNA synthetase (AOSC1122). Nas-13 belongs to a family of metalloproteinases involved in bone growth and morphogenesis (Xiong et al, 2006), and cysteinyltRNA synthetase regulates cysteine (cys) metabolism and protein synthesis (Hauenstein and Perona, 2008). Cys residues are common in many types of extracellular matrix proteins Pearsall et al, 2002) since disulfide bonds act to create a structural framework.…”