REGULATION OF PYRIDOXAL PHOSPHATE BIOSYNTHESIS IN E COLI

5‘-磷酸吡哆醛(PLP)是一种必需的、普遍存在的辅酶。 参与氨基酸和细胞代谢的许多方面。这个 这个持续项目的长期目标是确定PLP如何 生物合成在途径和基因水平上受到调节，并整合在一起 进入一般的细胞新陈代谢。E-Coli是一种理想的模型系统 这些生理、遗传和生化研究，因为它 合成一种形式的PLP前体，吡哆醇(PN；维生素B6)，如 植物和某些其他微生物，并能转化Pn，吡哆醛 (PL)和吡哆胺(PM)通过ALL中存在的清道夫途径转化为PLP 有机体。在这个五年计划中，我们将追求三个具体目标。 (一)我们将继续调查最重要的悬而未决问题 关于PLP生物合成的途径和生物化学。以下是 假说将会得到检验。(I)PLP需要EPD(GapB)基因 生物合成。(Ii)PLP生物合成缺失的分支包括 过多的转酮醇酶活性导致D-脱氧木糖的形成。 (3)纯化的SERC(PdxF)转氨酶使用两种不同的底物 导致PLP或L-丝氨酸的生物合成。(4)pdxA和pdxJ基因 产品封闭了PN的吡啶环。这一假设的推论是 PL/PM/PN激酶在清道夫中起作用，而不是在从头开始的PLP中起作用 生物合成途径。(V)PLP到PL的转换通过特定的 细胞内磷酸酶，类似于最近在哺乳动物中发现的那种。 (Ii)我们会继续发展基因研究方法，以研究 PLP生物合成途径的调节和功能。两个假设 将会受到考验。(I)在pdxB、SERC(PdxF)中检测到的替代分支，或 TktA、tktB突变体对新发PLP无显著影响 野生型细菌的生物合成。(Ii)缺乏途径的突变体和 可以分离出细胞内Pn和PLP的遗传调节 浓度增加，从而恢复突变体的功能 (PLP-KM)与PLP结合较差的酶。(三)我们会继续 研究该基因的结构、表达、调控和协调 PDX生物合成基因，所有这些基因都是复杂超操纵子的成员。 这一目标包括两个部分。(I)我们会研究该词的表达模式 将PDX基因作为一个群体来了解它们是否受到 常见机制，包括LRP、CRP和生长速度控制，或在 对某些生理条件的反应。(Ii)我们会研究某些 特定PDX基因调控中的有趣问题。这个 这个项目的继续很重要，有几个原因。它是 提供基本知识和对基本主题的见解 生物学兴趣，包括辅酶的调节和酶学 生物合成、复杂超操纵子的结构和调控、 生物合成途径的进化和重要类别的功能 酶的种类，如黄素蛋白氧化酶。这个项目已经导致了 关于整合辅酶的机制的重要新信息 生物合成为一般细胞新陈代谢。最后，这个项目是 生物医学和生物技术的相关性，因为PLP发挥得很好- 有案可查的，在人类中间代谢，生理， 和疾病。

Pyridoxal 5'-phosphate (PLP) is an essential, ubiquitous coenzyme that participates in many aspects of amino acid and cellular metabolism. The long term-goal of this continuing project is to determine how PLP biosynthesis is regulated at the pathway and genetic levels and integrated into general cellular metabolism. E coli is an ideal model system for these physiological, genetic, and biochemical studies, because it synthesizes a form of the PLP-precursor, pyridoxine (PN; vitamin B6), like plants and certain other microorganisms, and can convert PN, pyridoxal (PL), and pyridoxamine (PM) into PLP by a scavenger pathway present in all organisms. Three Specific Aims will be pursued in this five-year proposal. (I)We will continue to investigate the most important unresolved issues concerning the pathway and biochemistry of PLP biosynthesis. The following hypotheses will be tested. (i) The epd(gapB) gene is required for PLP biosynthesis. (ii) The missing branch of PLP biosynthesis involves redundant transketolase activities that lead to D-deoxyxylulose formation. (iii) Purified serC(pdxF) transaminase uses two different substrates leading to PLP or L-serine biosynthesis. (iv) The pdxA and pdxJ gene products close the pyridine ring of PN. A corollary of this hypothesis is that PL/PM/PN kinase functions in the scavenger and not the de novo PLP biosynthetic pathway. (v) Conversion of PLP to PL occurs by a specific intracellular phosphatase, analogous to the one recently found in mammals. (II) We will continue to develop genetic approaches to study the regulation and function of the PLP biosynthetic pathway. Two hypotheses will be tested. (i) An alternative branch detected in pdxB, serC(pdxF), or tktA tktB mutants does not contribute significantly to de novo PLP biosynthesis in wild-type bacteria. (ii) Mutants lacking pathway and genetic regulation can be isolated in which intracellular PN and PLP concentrations are increased, thereby restoring the functions of mutant (PLP-KM) enzymes that bind PLP poorly. (III) We will continue to investigate the structure, expression, regulation, and coordination of the pdx biosynthetic genes, all of which are members of complex superoperons. This Aim has two parts. (i) We will examine the expression patterns of the pdx genes as a group to learn whether they are coordinately regulated by common mechanisms, including LRP, CRP, and growth-rate control, or in response to certain physiological conditions. (ii) We will study certain interesting problems in the regulation of specific pdx genes. The continuation of this project is important for several reasons. It is providing basic knowledge and insights into topics of fundamental biological interest, including the regulation and enzymology of coenzyme biosynthesis, the structure and regulation of complex superoperons, the evolution of biosynthetic pathways, and the function of important classes of enzymes, such as flavoprotein oxidases. This project has led to significant new information about mechanisms that integrate coenzyme biosynthesis into general cellular metabolism. Finally, this project is of biomedical and biotechnological relevance, because PLP plays well- documented, diverse roles in human intermediary metabolism, physiology, and disease.