RESUMEN
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Mutación , Proteínas de Plantas/química , Ribulosa-Bifosfato Carboxilasa/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodopseudomonas/química , Rhodopseudomonas/enzimología , Rhodopseudomonas/genética , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spinacia oleracea/química , Spinacia oleracea/enzimología , Spinacia oleracea/genética , Relación Estructura-Actividad , Thermococcus/química , Thermococcus/enzimología , Thermococcus/genéticaRESUMEN
HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a K(d) value of 84 nM. ADP binds more tightly than does ATP, with a K(d) value of 15 nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Hidrogenasas/biosíntesis , Thermococcus/enzimología , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Sitios de Unión/genética , Cristalografía por Rayos X , Genes Arqueales , Hidrogenasas/química , Hidrogenasas/genética , Datos de Secuencia Molecular , Níquel/metabolismo , Thermococcus/genética , Thermococcus/crecimiento & desarrolloRESUMEN
Four virus-like integrated elements (TKV1, TKV2, TKV3, and TKV4) have been found in the genome of hyperthermophilic archaeon, Thermococcus kodakarensis, but virus particle formation has not been observed in the culture of T. kodakarensis. As the result of growth property analyses, mutants lacking each of the four virus-like regions exhibited decrease in the cell concentration and/or less growth rates compared to growth of parental strain (KU216), when the T. kodakarensis strains were grown at 85 °C in nutrient-rich medium. These results indicated that the genes in virus-like regions stimulated the cell growth under the observed growth condition. As the result of transcriptome analyses, genes involved in amino acid, energy or nucleotide metabolisms, and transport systems were up- or down-regulated in the cells of mutant strains. Interestingly, a decrease in transcriptional levels of glutamine synthetase (TK1796) gene (Tk-glnA) was observed in the cells of four mutant strains. Growths of TKV1 disrupted strain and TKV4 disrupted strain have shown no difference compared with that of KU216 by the addition of glutamate or glutamine, and the result suggested that TKV1 and TKV4 contributed to supply of amino acids to the cell.
