Homologs of aminoacyl-tRNA synthetases acylate carrier proteins and provide a link between ribosomal and nonribosomal peptide synthesis

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRSlike proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

Self-Referential Encoding on Modules of Anticodon Pairs—Roots of the Biological Flow System

“…It may be highly significant to the SRM propositions to find that also some of the known instances of non-ribosomal protein or peptide synthesis (NRPS) [111][112][113][114] Some proteins involved in NRPS show homologies to synthetases class II, especially the SerRS, but are small and monodomain, not bearing the tRNA binding activity while preserving the amino acid activation. Other instances of proteins active in NRPS show homologies to synthetases class I, closer to the Tyr-and TrpRS, and do not activate amino acids but utilize a couple of aminoacyl-tRNAs to promote sequential reactions for peptide synthesis and cyclo-dehydration.…”

Substrate Recognition by Novel Family of Amino Acid:[Carrier Protein] Ligases

“…The proteins were His-tagged at N-terminus and were purified by standard procedures on Ni-NTA agarose (Qiagen) as previously described. 21 In-frame fusions of N-terminal tRNA binding domain from Methanosarcina barkeri SerRS and aa:CP ligase coding sequences were created by overlap PCR. 21 PCR fragments coding for fusion proteins were cloned to pET28b, and N-terminal His-tagged fusion proteins were overexpressed and purified as described.…”

“…21 In-frame fusions of N-terminal tRNA binding domain from Methanosarcina barkeri SerRS and aa:CP ligase coding sequences were created by overlap PCR. 21 PCR fragments coding for fusion proteins were cloned to pET28b, and N-terminal His-tagged fusion proteins were overexpressed and purified as described. Preparation of SerRS from E. coli 22 and M. barkeri 23 was described previously.…”

“…In contrast, we have recently described and characterized a new class of truncated SerRS-like proteins, which activate selected amino acids, but are deprived of the tRNA-binding domain and thus they lack canonical tRNA aminoacylating activity. 21 Their genes were identified in numerous bacterial genomes. Expressed shortened SerRS-like proteins, which are structurally related to the catalytic core of atypical methanogenic-type SerRS (aSerRS), 21 were shown to transfer several activated amino acids to the -SH of phosphopantetheine prosthetic group, covalently attached to the carrier proteins of yet unknown function.…”

“…21 Their genes were identified in numerous bacterial genomes. Expressed shortened SerRS-like proteins, which are structurally related to the catalytic core of atypical methanogenic-type SerRS (aSerRS), 21 were shown to transfer several activated amino acids to the -SH of phosphopantetheine prosthetic group, covalently attached to the carrier proteins of yet unknown function. Therefore, these new enzymes act as amino acid:[carrier protein] ligases (aa:CP ligases).…”