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: 3304870
• 负责人: F ROBERT TABITA
• 金额: $ 16.57万
• 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/3304870
• 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; 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.

戊糖磷酸途径是一个重要的代谢方案所需的 几乎所有生物体的新陈代谢。 添加 两种独特的酶，二磷酸核酮糖羧化酶/加氧酶（RubisCO） 和磷酸核酮糖激酶（phosphoribulokinase，ERK），使这一途径的功能， 纯粹的生物合成模式，使生物体获得使用 二氧化碳是唯一的碳源。 在这种情况下， 该途径的其他酶，包括在细胞中发现的普遍存在的催化剂 原核生物和真核生物都是生物合成酶， 相反的方向形成它们在体内的通常作用。 我们实验室最近的研究表明， 上述酶，沿着编码果糖二磷酸酶的基因 磷酸甘油醛脱氢酶和转酮醇酶， 发现于厌氧细胞的两个不同的染色体操纵子中 紫色非硫细菌类球红杆菌。 两种重组 大肠杆菌中的蛋白质，以及许多重组蛋白质 纯化至均一。 因此，该项目的主要目标之一是 研究了β-淀粉样蛋白、FBPase和 转酮醇酶，使用蛋白质化学和分子生物学技术， 生物学，这种双重方法第一次变得可行， 这些重要的蛋白质。 该项目的第二个主要目标是研究 两个操纵子的差异调节的基础。 控制 表现在转录和转录后水平。 由于暴露在碳和氧含量增加或减少的环境中 对基因表达有重大影响，我们将探讨 控制及其与细胞内调节系统的关系， 控制机制及其与细胞内调节系统的关系 对外界刺激有反应 这项研究还关注 多顺反子转录物的溶核加工的作用， 它与差异基因表达的关系， 调节原核和真核基因的表达。 因此，这些研究提供了一个极好的机会， 在酶和分子水平上， 了解中间代谢。