RESUMEN
Accurate protein synthesis requires the hydrolytic editing of tRNAs incorrectly aminoacylated by aminoacyl-tRNA synthetases (ARSs). Recognition of cognate tRNAs by ARS is less error-prone than amino acid recognition, and, consequently, editing domains are generally believed to act only on the tRNAs cognate to their related ARSs. For example, the AlaX family of editing domains, including the editing domain of alanyl-tRNA synthetase and the related free-standing trans-editing AlaX enzymes, are thought to specifically act on tRNA(Ala), whereas the editing domains of threonyl-tRNA synthetases are specific for tRNA(Thr). Here we show that, contrary to this belief, AlaX-S, the smallest of the extant AlaX enzymes, deacylates Ser-tRNA(Thr) in addition to Ser-tRNA(Ala) and that a single residue is important to determine this behavior. Our data indicate that promiscuous forms of AlaX are ancestral to tRNA-specific AlaXs. We propose that former AlaX domains were used to maintain translational fidelity in earlier stages of genetic code evolution when mis-serylation of several tRNAs was possible.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Arqueales/metabolismo , Código Genético , Pyrococcus abyssi/metabolismo , Pyrococcus horikoshii/metabolismo , ARN de Transferencia/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Pyrococcus abyssi/clasificación , Pyrococcus abyssi/genética , Pyrococcus horikoshii/clasificación , Pyrococcus horikoshii/genética , Edición de ARN , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes. In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.
Asunto(s)
Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Expresión Génica , Glutamato Sintasa/genética , Glutamato Sintasa/metabolismo , Pyrococcus horikoshii/enzimología , Proteínas Arqueales/química , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato Sintasa/química , Cinética , Datos de Secuencia Molecular , Filogenia , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pyrococcus horikoshii/química , Pyrococcus horikoshii/clasificación , Pyrococcus horikoshii/genéticaRESUMEN
BACKGROUND: Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to determine the function of the hypothetical proteins. RESULTS: We examined 960 soluble proteins from Pyrococcus and selected 107 interactions based on luciferase reporter activity, which was then evaluated using a computational approach to assess the reliability of the interactions. We also analyzed the expression of the assay samples by western blot, and a few interactions by in vitro pull-down assays. We identified 11 hetero-interactions that we considered to be located at the same operon, as observed in Helicobacter pylori. We annotated and classified proteins in the selected interactions according to their orthologous proteins. Many enzyme proteins showed self-interactions, similar to those seen in other organisms. CONCLUSION: We found 13 unannotated proteins that interacted with annotated proteins; this information is useful for predicting the functions of the hypothetical Pyrococcus proteins from the annotations of their interacting partners. Among the heterogeneous interactions, proteins were more likely to interact with proteins within the same ortholog class than with proteins of different classes. The analysis described here can provide global insights into the biological features of the protein-protein interactions in P. horikoshii.