RESUMO
The crystal structure of PurL from Thermus thermophilus HB8 (TtPurL; TTHA1519) was determined in complex with an adenine nucleotide, PO(4)(3-) and Mg(2+) at 2.35 Å resolution. TtPurL consists of 29 α-helices and 28 ß-strands, and one loop is disordered. TtPurL consists of four domains, A1, A2, B1 and B2, and the structures of the A1-B1 and A2-B2 domains were almost identical to each other. Although the sequence identity between TtPurL and PurL from Thermotoga maritima (TmPurL) is higher than that between TtPurL and the PurL domain of the large PurL from Salmonella typhimurium (StPurL), the secondary structure of TtPurL is much more similar to that of StPurL than to that of TmPurL.
Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Thermus thermophilus/enzimologia , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Domínio Catalítico , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
An alcohol dehydrogenase (ADH) gene, ST0053, from Sulfolobus tokodaii was expressed in Escherichia coli. The purified recombinant enzyme was an NAD-dependent medium-chain ADH with high thermostability and tolerance of a wide range of pHs. This is the first step in creating an experimental functionality library of 10 genes annotated as ADHs in the S. tokodaii genome.
Assuntos
Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Sulfolobus/enzimologia , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Clonagem Molecular , Coenzimas/farmacologia , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , NAD/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sulfolobus/genéticaRESUMO
The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.
Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/química , Methanocaldococcus/química , Sulfolobus/química , Thermus thermophilus/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Methanocaldococcus/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologia , Thermus thermophilus/enzimologiaRESUMO
Glycinamide ribonucleotide synthetase (GAR-syn, PurD) catalyses the second reaction of the purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine and ATP to glycinamide ribonucleotide (GAR), ADP and Pi. In the present study, crystal structures of GAR-syn's from Thermus thermophilus, Geobacillus kaustophilus and Aquifex aeolicus were determined in apo forms. Crystal structures in ligand-bound forms were also determined for G. kaustophilus and A. aeolicus proteins. In general, overall structures of GAR-syn's are similar to each other. However, the orientations of the B domains are varied among GAR-syn's and the MD simulation suggested the mobility of the B domain. Furthermore, it was demonstrated that the B loop in the B domain fixes the position of the ß- and γ- phosphate groups of the bound ATP. The structures of GAR-syn's and the bound ligands were compared with each other in detail, and structures of GAR-syn's with full ligands, as well as the possible reaction mechanism, were proposed.