| dc.description.abstract | Aminoacyl-tRNA synthetases (aaRSs) belongs to a group of enzymes necessary for protein synthesis. Their main function is to attach an amino acid to its corresponding tRNA to form aminoacyl-tRNA, which is then brought to the ribosome for protein synthesis. One aaRSs corresponds to each amino acid. Previous studies have shown that although some bacteria lack CysRS, they can synthesize Cys-tRNACys through an indirect pathway using serine as a substrate, whereas most bacteria have a CysRS to synthesize Cys-tRNACys. Herein, we present the evidence that that Mycobacterium abscessus possesses two cysteinyl-tRNA synthetase (CysRS) homologues genes that CysS1 and CysS2 (which encode MaCysRS1 and MaCysRS2, respectively). Two homologous CysRSs in M. abscessus have a 37% identity, 80% similarity and 37-42% identity with E. coli CysRS, it is totally different with CysRS in Escherichia coli. Further sequence and phylogenetic analyses showed that MaCysRS2 is actually a mycothiol cysteine-ligase (MshC) which is involved in Mycothiol (MSH) synthesis as a protective thiol. It is not only different protein involved in protein synthesis but also lacks anticodon-binding domain. The result of complementation assay showed that both MaCysS1 and MaCysS2 were moderately expressed in the yeast but failed to complement the cytoplasmic function of the knockout strain, i.e., these two genes cannot provide the required CysRS activity to support the growth of the null allele on 5’-FOA medium. However, if a mitochondrial targeting signal (MTS) was attached to the N-terminal of the MaCysRS1, the fusion protein successfully rescued the growth defect of the knockout strain on YPG, suggesting that this fusion protein can substitute the mitochondrial activity of yeast CysRS. In contrast, MaMshC, even fused to an MTS, could not do so, probably because MaMshC lacks an anticodon-binding domain (ABD). Most surprisingly, fusion of a tRNA-binding domain of Arc1p to MTS-MaMshC, yielding an MTS-MaMshC-Arc1p (M+C), enabled the enzyme to restore the growth of the yeast knockout strain on YPG. This result shows that MaMshC, a bacterial protective thiol-producing enzyme, can be converted to a functional cysteinyl-tRNA synthetase through fusion of a non-specific tRNA-binding domain.
Keywords: CysRS, MaCysRS, Arc1p
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