MOLECULAR BASIS AND ENZYMOLOGY OF MICROBIAL BIOSYNTHESIS

微生物生物合成的分子基础和酶学

基本信息

批准号：
2415149
负责人：
F ROBERT TABITA
金额：
$ 30.26万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-05-01 至 2000-04-30
项目状态：
已结题

项目摘要

Normal and healthy cellular activity is dependent on a concerted interplay of genetic regulation and protein function. Our primary interest and long- term goal is to understand genetic and biochemical factors which influence the function of an important metabolic route, the pentose phosphate pathway. This is a metabolic scheme required for the metabolism of virtually all living organisms. With the addition of two unique enzymes, ribulose bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), this pathway functions 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 such as transkelotase (TK) and fructose l ,6-bisphosphatase (FBPase), found in both prokaryotes and eukaryotes, function as biosynthetic enzymes, in the opposite direction from their usual role in vivo. The genes encoding nearly all the enzymes of this pathway have been isolated and found to be associated in distinct chromosomal operons (the cbb regulon) in the nonsulfur purple bacterium Rhodobacter sphaeroides. In addition, a transcriptional activator protein and a gene that encodes a sensor kinase, have been found to regulate the expression of the cbb genes. Other regulatory elements have also been discovered and much of this study is directed at elucidating the regulatory mechanism that mitigates the sensory transduction pathway controlling gene expression and ultimately biosynthetic metabolism. Since exposure to varying levels of carbon and oxygen has a profound effect on gene expression, a concerted effort will focus on relating such external stimuli to the regulatory cascade. The second major thrust of this project will involve a study of structure- function relationships of PRK, FBPase, and TK. Since their genes have been expressed as highly active recombinant proteins that are easily purified, it will be possible to use site-directed mutagenesis procedures in combination with known x-ray structural models to enhance our knowledge of how these proteins function. These are all extremely important metabolic enzymes; for example TK is required for thiamine metabolism in all cells and alteration of its kinetic and chemical properties leads to severe pathological conditions including nutritional deficiency, alcoholism, Wernicke-Korsakoff encephalopathy, and Alzheimer's disease. FBPase is essential for gluconeogenesis and PRO is one of the enzymes unique to the reductive pentose phosphate pathway. In each case, the recombinant systems developed here have the potential to substantially increase available information of these enzymes thus affording an unusual opportunity to relate the control of cellular metabolism to the function and structure of key catalysts.

正常和健康的细胞活性取决于协同的相互作用遗传调节和蛋白质功能。我们的主要兴趣和长期术语目标是了解影响影响的遗传和生化因素重要的代谢途径的功能，五肽磷酸盐路径。这是代谢的代谢方案几乎所有的生物体。添加了两个独特的酶，核糖双磷酸羧化酶/氧合酶（Rubisco）和磷酸胰蛋白酶（PRK），该途径在纯生物合成中起作用模式，使生物具有使用二氧化碳作为唯一的碳来源。在这些条件下，其他酶途径，包括无处不在的催化剂，例如transkelotase（TK）和果糖L，6-二磷酸酶（FBPase），在原核生物和真核生物在相反方向上充当生物合成酶从他们在体内的通常角色。编码几乎所有酶的基因该途径已被隔离并发现与众不同非硫紫色细菌中的染色体操纵子（CBB调节子） Rhodobacter sphaeroides。另外，转录激活蛋白并且已经发现一个编码传感器激酶的基因来调节 CBB基因的表达。其他监管元素也是发现的，这项研究的大部分是针对阐明减轻感觉转导途径的调节机制控制基因表达并最终生物合成代谢。自从暴露于不同水平的碳和氧气对基因表达，一致的努力将集中于关联此类外部刺激调节级联。该项目的第二个主要力量将涉及对结构的研究 - PRK，FBPase和TK的功能关系。因为他们的基因有被表示为高度活性的重组蛋白，很容易纯化，可以使用定位的诱变程序结合已知的X射线结构模型，以增强我们的知识这些蛋白质如何发挥作用。这些都非常重要代谢酶；例如，硫胺素代谢需要TK 所有细胞及其动力和化学特性的改变导致严重的病理状况，包括营养缺乏，酒精中毒，Wernicke-Korsakoff脑病和阿尔茨海默氏病。 FBPase对于糖生成至关重要，Pro是酶之一还原性戊糖磷酸途径独有。在每种情况下，这里开发的重组系统有可能实质上增加这些酶的可用信息，从而提供不寻常的信息将细胞代谢控制与功能联系起来的机会和关键催化剂的结构。