REGULATION OF PYRIDOXAL PHOSPHATE BIOSYNTHESIS IN E COLI

这个持续项目的长期目标是学习如何 吡哆醇(维生素B6；PN)和磷酸吡哆醛(PLP)的生物合成 在途径和基因水平上受到调控，并整合到一般 细胞新陈代谢。吡哆醛磷酸盐是一种基本的，普遍存在的 辅酶在细胞新陈代谢中扮演多种角色的辅酶，尤其是 氨基酸，存在于所有有机体中。相当多的证据表明，PN和 PLP浓度在途径和基因上受到严格调控 水平；然而，关于这种机制的证明相对较少 带来这一规定。在这方面，大肠杆菌是一种理想的 这些基础广泛的生理、遗传和 生物化学研究，因为它合成了PN，就像植物和某些 其他微生物，然后通过两步途径将PN转化为PLP 这似乎是普遍现象。 这项五年计划有四个具体目标。第一, 对Pn生物合成的调节将继续进行研究。目标 包括PdxB分析和血清分子进化、生化 PDX酶功能的表征，继续分子遗传学 有趣的PDX复合体的结构与调控分析 操纵子，分离PDX的有前景的遗传方法的发展 调节突变体，以及一条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，从Pn到Pn的普遍途径的调节 PLP将得到进一步的研究。AIM II包括识别、测绘、 必需的pdxK(PN/PL激酶)的分离及其调控分析 基因和pdxT(PN/PL促进剂)和pdxD(PL脱氢酶)基因， 关于净光合速率调节的几个假说的批判性评价 PdxH的PNP、PLP途径及其遗传和酶学特征 (PNP氧化酶)。第三，B6维生素和PLP水平将在PDX中进行测量 突变体和在各种压力条件下生长的细菌， 包括那些引起PLP需要酶的大量诱导的酶。 第四，PN/PL促进者的特征将是了解E. 大肠杆菌使用孔或载体来扩散环状化合物。 这个项目的继续是重要的，有几个原因。它 提供有关辅酶生物合成调控的基本知识。 一种生理上和基因上容易驯服的有机体。当前的实验 即将对几个关键假设进行关键测试，这些假设 PLP的生物合成受到调控。该项目提供了重要的 关于将辅酶生物合成整合到 一般细胞新陈代谢。该项目的几个方面超越了 辅酶生物合成，并提供了对基础主题的见解 生物兴趣，如复合体的结构和调节 操纵子与生物合成途径的进化。最后，增加了 关于PLP生物合成的知识具有明显的生物医学和 生物技术的相关性，因为PLP发挥着有据可查、多样化的作用 在人类中间代谢、生理和疾病中的作用。

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.