MOLECULAR BASIS AND ENZYMOLOGY OF MICROBIAL BIOSYNTHESIS

微生物生物合成的分子基础和酶学

• 批准号: 2183145
• 负责人: F ROBERT TABITA
• 金额: $ 17.76万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183145
• 关键词: Rhodospirillales; affinity labeling; bacterial genetics; fructose biphosphatase; gene deletion mutation; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; microorganism metabolism; nucleic acid sequence; operon; pentose phosphate shunt; phosphorylase kinase; protein structure function; recombinant DNA; recombinant proteins; site directed mutagenesis; structural genes; transaldolase /transketolase

The pentose phosphate pathway is an important metabolic scheme required for the metabolism of virtually all living organisms. The addition of two unique enzymes, ribulose bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), allows this pathway to function in a purely biosynthetic mode, such that organisms gain the capacity to use carbon dioxide as the sole source of carbon. Under these conditions, other enzymes of the pathway, including ubiquitous catalysts found in both prokaryotes and eukaryotes, function as biosynthetic enzymes, in the opposite direction form their usual role in vivo. Recent work from our laboratory has shown that the structural genes for the above enzymes, along with genes encoding fructose bisphosphatase (FBPase), glyceraldehyde phosphate dehydrogenase, and transketolase, are found in two distinct chromosomal operons in the facultatively anaerobic purple nonsulfur bacterium Rhodobacter sphaeroides. Both recombinant proteins in Escherichia coli, and many of the recombinant proteins purified to homogeneity. Thus, one of the major goals of this project is to study structure-function relationships of PRK, FBPase and transketolase, using the techniques of protein chemistry and molecular biology, the first time that this dual approach has become feasible for these important proteins. The second major thrust of this project will be to study the molecular basis for differential regulation of the two operons. Control is manifested at both the transcriptional and posttranscriptional levels. Since exposure to increasing or decreasing levels of carbon and oxygen has a major effect on gene expression, we will probe the mechanism of control and its relation to intracellular regulatory systems known to be mechanism of control and its relation to intracellular regulatory systems known to be responsive to external stimuli. This research also focuses on the role of nucleolytic processing of polycistronic transcripts and its relation to differential gene expression, a key factor important for regulating expression of both prokaryotic and eukaryotic genes. These studies thus present an excellent opportunity to relate control at both the enzymic and molecular levels, to increase our general understanding of intermediary metabolism.

戊糖磷酸盐途径是必需的重要代谢方案 对于几乎所有生物体的代谢。 添加 两种独特的酶，核糖双磷酸羧化酶/氧合酶（Rubisco） 和磷酸胰蛋白酶（PRK）允许此途径在A中发挥作用 纯粹的生物合成模式，使生物具有使用能力 二氧化碳作为唯一的碳源。 在这些条件下， 途径的其他酶，包括在 原核生物和真核生物都充当生物合成酶，在 相反的方向在体内构成了通常的角色。 我们实验室的最新工作表明 上述酶以及编码果糖双磷酸酶的基因 （FBPase），磷酸甘油醛脱氢酶和转酮醇酶是 在辅助厌氧中的两个不同的染色体操纵子中发现 紫色非硫细菌杜鹃花杆菌。 都是重组 大肠杆菌中的蛋白质和许多重组蛋白 纯化为同质性。 因此，该项目的主要目标之一是 研究PRK，FBPase和 转酮醇，使用蛋白质化学技术和分子技术 生物学，这种双重方法首次变得可行 这些重要的蛋白质。 该项目的第二个主要力量是研究分子 对两个操纵子的差异调节的基础。 控制是 在转录和转录后水平上都表现出来。 由于暴露于碳和氧气水平的增加或降低 对基因表达有重大影响，我们将探究 控制及其与已知为的细胞内调节系统的关系 控制机理及其与细胞内调节系统的关系 已知对外部刺激有反应。 这项研究也重点 关于多水分转录本和核酸化处理的作用 它与差异基因表达的关系，这对于 调节原核生物和真核基因的表达。 因此，这些研究为关联控制的绝佳机会 酶和分子水平都增加了我们的一般 了解中介代谢。