RESUMEN
The hyperthermophilic archaeon, Sulfolobus, synthesizes lysine via the α-aminoadipate pathway; however, the gene encoding homocitrate synthase, the enzyme responsible for the first and committed step of the pathway, has not yet been identified. In the present study, we identified saci_1304 as the gene encoding a novel type of homocitrate synthase fused with a Regulation of Amino acid Metabolism (RAM) domain at the C terminus in Sulfolobus acidocaldarius. Enzymatic characterization revealed that Sulfolobus homocitrate synthase was inhibited by lysine; however, the mutant enzyme lacking the RAM domain was insensitive to inhibition by lysine. The present results indicated that the RAM domain is responsible for enzyme inhibition.
Asunto(s)
Proteínas Arqueales/metabolismo , Oxo-Ácido-Liasas/metabolismo , Sulfolobus acidocaldarius/enzimología , Proteínas Arqueales/química , Proteínas Arqueales/genética , Sitios de Unión , Lisina/metabolismo , Mutación , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/genética , Unión ProteicaRESUMEN
Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two ß-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg2+, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/genética , Proteínas Bacterianas/genética , Isocitrato Deshidrogenasa/genética , Sulfolobus acidocaldarius/enzimología , 3-Isopropilmalato Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/metabolismo , Magnesio/metabolismo , Malatos/metabolismo , Unión Proteica , Sulfolobus acidocaldarius/genéticaRESUMEN
Matriptase/MT-SP1, a type II membrane serine protease widely expressed in normal epithelial cells and human carcinoma cells, is thought to be involved in cancer progression. To clarify this possibility, we overexpressed exogenous matriptase in the human stomach cancer cell line AZ521. In vitro, the matriptase transfectant (Mat-AZ521) and the control transfectant (Mock-AZ521) showed a similar growth rate, although the saturation cell density was significantly higher with the Mat-AZ521. When implanted into nude mice subcutaneously or intraperitoneally, Mat-AZ521 cells grew faster and produced much larger solid tumors than Mock-AZ521 cells. The overexpression of matriptase in AZ521 cells shortened the survival time of tumor-bearing mice. Histological analysis showed that both the number and the size of blood vessels in tumor tissues were significantly higher in the Mat-AZ521 tumors than the Mock-AZ521 ones. Moreover, it was found that purified matriptase activated one of the important matrix metalloproteinases, stromelysin (MMP-3). These results suggest the possibility that the matriptase-dependent activation of MMP-3, as well as the direct activity of matriptase, promotes tumor growth and angiogenesis by enhancing extracellular matrix degradation in tumor cell microenvironments.