REGULATION OF PYRIDOXAL PHOSPHATE BIOSYNTHESIS IN E COLI

大肠杆菌中磷酸吡哆醛生物合成的调控

• 批准号: 2178828
• 负责人: MALCOLM E. WINKLER
• 金额: $ 20.17万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178828
• 关键词: Escherichia coli; aminoacid metabolism; bacterial genetics; biochemical evolution; chimeric proteins; cofactor; gene expression; genetic mapping; immunoprecipitation; mutant; nucleic acid sequence; nutrition related tag; operon; pore forming protein; protein biosynthesis; pyridoxal phosphate; pyridoxine; transposon /insertion element; vitamin biosynthesis

The long term goal of this continuing project is to learn how the biosynthesis of pyridoxine (vitamin B6;PN) and pyridoxal phosphate (PLP) are regulated at the pathway and genetic levels and integrated into general cellular metabolism. Pyridoxal phosphate is an essential, ubiquitous coenzyme that plays numerous roles in cellular metabolism, particularly of amino acids, in all organisms. Considerable evidence suggests that PN and PLP concentrations are strictly regulated at the pathway and genetic levels; yet, relatively little has been proven about the mechanisms that bring about this regulation. In this regard, Escherichia coli is an ideal model organism for these broadly based physiological, genetic, and biochemical studies, because it synthesizes PN, like plants and certain other microorganisms, and then converts PN into PLP by a two-step pathway that seems to be universal. Four Specific Aims are planned for this five-year proposal. First, investigation will be continued of the regulation of PN biosynthesis. Aim I includes analysis of PdxB and SerA molecular evolution, biochemical characterization of Pdx enzyme functions, continued molecular genetic analyses of the structure and regulation of interesting pdx complex operons, development of promising genetic approaches to isolate pdx regulatory mutants, and genetic and biochemical examination of a pathway that may provide a shared precursor for vitamins B1, B2, and B6 biosynthesis. Second, the regulation of the universal pathway from PN to PLP will be studied further. Aim II includes identification, mapping, isolation, and regulatory analyses of the essential pdxK (PN/PL kinase) gene and the pdxT (PN/PL facilitator), and pdxD (PL dehydrogenase) genes, critical evaluation of several hypotheses about the regulation of the PN PNP PLP pathway, and genetic and enzymological characterization of pdxH (PNP oxidase). Third, B6 vitamer and PLP levels will be measured in pdx mutants and in bacteria grown under a variety of stress conditions, including ones that cause large inductions of PLP-requiring enzymes. Fourth, the PN/PL facilitator will be characterized to learn whether E. coli uses a pore or carrier for diffusion of ringed compounds. The continuation of this project is important for several reasons. It provides basic knowledge about the regulation of coenzyme biosynthesis in a physiologically and genetically tractable organism. Current experiments are at the point of critically testing several key hypotheses about how PN and PLP biosynthesis is regulated. The project is providing significant new information about mechanisms that integrate coenzyme biosynthesis into general cellular metabolism. Several aspects of the project transcend coenzyme biosynthesis and are offering insights into topics of fundamental biological interest, such as the structure and regulation of complex operons and the evolution of biosynthetic pathways. Finally, increased knowledge about PLP biosynthesis is of obvious biomedical and biotechnological relevance, because PLP plays well-documented, diverse roles in human intermediary metabolism, physiology, and disease.

这个持续项目的长期目标是了解 吡ido醇（维生素B6; PN）和吡啶还毒素（PLP）的生物合成（PLP） 在途径和遗传水平上进行调节，并整合到一般 细胞代谢。 吡啶还毒磷酸是必不可少的，无处不在的 辅酶在细胞代谢中起许多作用，特别是 在所有生物中，氨基酸。 大量证据表明PN和 PLP浓度受到严格调节的途径和遗传 水平；然而，关于机制的证明相对较少 提出这一法规。 在这方面，大肠杆菌是理想的 这些基于这些广泛的生理，遗传和 生化研究，因为它合成了PN，例如植物和某些 其他微生物，然后通过两步途径将PN转换为PLP 这似乎是普遍的。 计划为期五年的提案计划四个具体目标。 第一的， 将继续调查PN生物合成。 目的 我包括PDXB和血清分子进化的分析，生化 PDX酶功能的表征，持续的分子遗传 对有趣的PDX复合物的结构和调节的分析 操纵子，开发有希望的遗传方法来分离PDX 调节突变体以及途径的遗传和生化检查 这可能为维生素B1，B2和B6提供共同的前体 生物合成。 第二，通用途径从PN到 PLP将进一步研究。 AIM II包括标识，映射， 基本PDXK（PN/PL激酶）的分离和调节分析 基因和PDXT（PN/PL促进剂）和PDXD（PL脱氢酶）基因， 对PN调节的几种假设的批判性评估 PNP PLP途径以及PDXH的遗传和酶学特征 （PNP氧化酶）。 第三，将在PDX中测量B6 Vitamer和PLP水平 突变体和在多种应力条件下生长的细菌中， 包括引起大量诱导PLP的酶的酶。 第四，PN/PL促进器将被表征以了解E. E.是否。 大肠杆菌使用孔或载体来扩散环形化合物。 由于几个原因，该项目的延续很重要。 它 提供有关调节辅酶生物合成的基本知识 一种生理和遗传性的生物体。 当前的实验 正在批判性地测试有关PN的几个关键假设 调节PLP生物合成。 该项目提供了重要的 有关将辅酶生物合成整合到中的机制的新信息 一般细胞代谢。 该项目的几个方面超越 辅酶生物合成，并为基本主题提供见解 生物学兴趣，例如复杂的结构和调节 操纵子和生物合成途径的进化。 最后，增加了 关于PLP生物合成的知识是显而易见的生物医学和 生物技术相关性，因为PLP扮演着据可查，多样 人类中介代谢，生理和疾病中的作用。