Naslov
Homologs of class II aminoacyl-tRNA synthetases: a possible link between protein synthesis and non-ribosomal peptide synthesis

Autori
Močibob, Marko ; Ivić, Nives ; Bilokapić, Silvija ; Maier, Timm ; Luić, Marija ; Ban, Nenad ; Weygand-Đurašević, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
23rd tRNA Workshop: From origin of life to biomedicine / Weil, Tobias ; Santos, Manuel A. S. - Aveiro : University of Aveiro, 2010

Skup
23rd tRNA Workshop

Mjesto i datum
Aveiro, Portugal, 28.01.2010. - 02.02.2010

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
aminoacylation ; seryl-tRNA synthetase ; non-canonical functions of aaRS ; amino acid:[carrier protein] ligase (AMP-forming)

Sažetak
Analysis of the completed genome sequences revealed the presence in various bacteria (predominantly α- and β-proteobacteria) of an open reading frame encoding a polypeptide homologous to the catalytic domain of atypical methanogenic-type seryl-tRNA synthetases, but lacking the N-terminal tRNA binding domain. All three signature motifs of class II aminoacyl- tRNA synthetases are preserved in the sequence of truncated SerRS homologs, as well as the residues corresponding to those that coordinate the active site zinc ion, required for serine binding in methanogenic-type SerRSs. Three representatives of truncated SerRS homologs were cloned and purified for biochemical and structural characterization: two from Bradyrhizobium japonicum and one from Agrobacterium tumefaciens. As expected, they all formed homodimers. Surprisingly, the two B. japonicum SerRS-like enzymes activate glycine, not serine, while the homolog from A. tumefaciens is more promiscuous and preferentially activates alanine, and to lesser extent glycine and serine. B. japonicum SerRS- like protein was crystallized in free form and complexed with ATP and the non-hydrolysable analog of glycyl-adenylate. The structures revealed a remarkable resemblance to catalytic core of methanogenic-type SerRS, with ATP bound in bent conformation and the zinc-ion participating in coordination of glycine amino- group. Enzymes were tested for tRNA- aminoacylation activity using bulk tRNA isolated from their native sources. No such activity could be detected, even when the tRNA- binding domain from Methanosarcina barkeri SerRS was fused to recombinant proteins. Curiously, enzymes were capable of hydrolyzing ATP in the presence of specific amino acids and different thiols (cysteine, ditiothreitol, coenzyme A), and transferring the amino acid to respective -SH groups. This propensity, together with a strong synteny of novel SerRS homologs with the genes for putative phosphopantetheine binding proteins in a number of sequenced genomes, prompted us to clone and express the carrier proteins and test them as the substrates for aminoacylation. After attachment of phosphopantetheine prosthetic arm, the carrier proteins were efficiently charged with respective amino acids. The transfer of activated amino acids specifically to the phosphopantetheine prosthetic group was confirmed by mass spectrometry and chemical labeling. Kinetic analysis of thioester bond formation catalyzed by the truncated SerRS homologs revealed the parameters comparable to tRNA aminoacylation by canonical aaRSs. The fate of amino acid transferred to carrier protein is not clear yet, but it is obviously diverted from protein biosynthesis. Our data suggest that homologs of class II aaRS represent a new type of amino acid:[carrier protein] ligases (AMP-forming), possibly providing an exciting link between ancient thioester world of non-ribosomal peptide synthesis and contemporary protein biosynthesis.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA