REGULATION OF PYRIDOXAL PHOSPHATE BIOSYNTHESIS IN E COLI

这个持续项目的长期目标是学习如何 维生素B6和磷酸吡哆醛的生物合成 在途径和遗传水平上受到调节，并整合到一般的 细胞代谢 磷酸吡哆醛是一种必需的，普遍存在的 辅酶，在细胞代谢中起着许多作用，特别是 氨基酸，在所有生物体中。 大量证据表明，PN和 PLP浓度在途径和遗传上受到严格调节。 水平;然而，相对较少的是已经证明的机制， 促成了这一规定。 在这方面，大肠杆菌是理想的 模式生物为这些广泛基础的生理，遗传， 生化研究，因为它合成PN，像植物和某些 其它微生物，然后通过两步途径将PN转化为PLP 这似乎是普遍的。 这一五年计划有四个具体目标。 第一、 将继续研究PN生物合成的调节。 目的 I包括PdxB和SerA的分子进化分析，生物化学分析， Pdx酶功能表征、持续分子遗传学 有趣的pdx复合物的结构和调控分析 操纵子，开发有前途的遗传方法分离pdx 调节突变体，以及途径的遗传和生物化学检查 可以提供维生素B1、B2和B6的共同前体 生物合成 第二，从PN到 将进一步研究PLP。 目标二包括识别、绘图、 分离，并对必需的pdxK（PN/PL激酶）进行调控分析 基因和pdxT（PN/PL促进剂）和pdxD（PL脱氢酶）基因， 关于PN调节的几个假设的批判性评价 PNP PLP途径，以及pdxH的遗传和酶学表征 (PNP氧化酶）。 第三，将在pdx中测量B6维生素和PLP水平 突变体和在各种应激条件下生长的细菌中， 包括引起大量诱导需要PLP的酶的那些。 第四，PN/PL主持人将被定性，以了解E。 大肠杆菌使用孔或载体来扩散环状化合物。 由于几个原因，该项目的继续是重要的。 它 提供了关于辅酶生物合成调节的基本知识， 生理学和遗传学上易于控制的有机体。 当前实验 目前正在严格测试几个关键假设， 并且PLP生物合成受到调节。 该项目提供了重要的 新的信息机制，整合辅酶生物合成， 一般细胞代谢。 该项目的几个方面超越了 辅酶生物合成，并提供见解的主题，基本 生物学兴趣，如复合物的结构和调控 操纵子和生物合成途径的进化。 最后，增加 关于PLP生物合成知识具有明显的生物医学意义， 生物技术的相关性，因为PLP发挥良好的记录，多样性， 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.