http___fulir.irb.hr_791_.xml
http___fulir.irb.hr_2150__1.xml
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Močibob, Marko; Ivić, Nives; Šubašić, Deni; Luić, Marija; Weygand-Đurašević, Ivana (2011) Substrate Recognition by Novel Family of Amino Acid:[Carrier Protein] Ligases. Croatica Chemica Acta, 84 (2). Abstract Amino acid:[carrier protein] ligases (aa:CP ligases) are newly discovered homologs of aminoacyl-tRNA synthetases (aaRS) which aminoacylate carrier proteins instead of tRNA. Activated amino acids are attached to prosthetic group of carrier proteins by thioester bond. Weak thioacylation activity was observed for many conventional aaRS. Therefore, different thiols were investigated as substrate analogs for aa:CP ligases. Here we show that coenzyme A, dithiothreitol and cysteine are efficiently aminoacylated by aa:CP ligases. The crystal structure of aa:CP ligase from Bradyrhizobium japonicum in complex with coenzyme A was solved, and revealed that CoA, besides acting as the substrate analog of the carrier protein, also competes with ATP for binding to the active site. aa:CP ligases do not aminoacylate tRNA, although they remarkably resemble catalytic core of atypical seryl-tRNA synthetases (aSerRS) from methanogenic archaea. Since aa:CP ligases lack tRNA-binding domain, fusion proteins of aa:CP ligases and aSerRS tRNA-binding domain were prepared in attempt to restore tRNA aminoacylation activity. Although fusion proteins were able to bind tRNA through appended domain, tRNA could not substitute carrier proteins in aminoacylation reaction.
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Ivić, Nives (2013) Structural characterization of bacterial glycine:[carrier protein] ligase and its complexes with substrates. Doctoral thesis, Sveučilište u Zagrebu, Prirodoslovno-matematički fakultet. Abstract Nedavno je otkrivena nova porodica enzima sličnih metanogenom tipu seril-tRNA-sintetaza. Nazvani aminokiselina:[protein-nosač]-ligaze, ovi homolozi umjesto serina tioesterski vežu glicin ili alanin na odgovarajući protein-nosač (CP) umjesto na tRNA. U ovom radu je strukturno okarakteriziran član te porodice ; glicin:[protein-nosač]-ligaza 1 (Bj Gly:CP-ligaza 1) iz bakterije Bradyrhizobium japonicum. Usporedba kristalnih struktura Bj Gly:CP-ligaze 1 i metanogenog tipa seril-tRNA-sintetaze je otkrila strukturnu očuvanost katalitičke domene i aktivnog mjesta enzima. Strukture Bj Gly:CP-ligaze 1 u kompleksu s malim supstratima (ATP-om i analogom glicil-adenilata) su ukazale na potencijalne aminokiseline odgovorne za diskriminaciju glicina i očuvanost mehanizma aktivacije glicina. Analiza strukture kompleksa Bj Gly:CP-ligaze 1 i odgovarajućeg CP-a je otkrila međudjelovanje proteina isključivo preko CP-vezne zavojnice karakteristične za aminokiselina:[protein-nosač]-ligaze. Dizajnirana je hibridna Gly:CP-ligaza u kojoj je CP-vezna zavojnica iz Bj Gly:CP-ligaze 1 zamijenjena odgovarajućom iz Agrobacterium tumefaciens alanin:[protein-nosač]-ligaze. Usporedbom kompleksa ligaza s odgovarajućim CP-om razjašnjena je strukturna osnova na temelju koje aa:CP-ligaze razlikuju protein-nosače.