MOLECULAR BASIS AND ENZYMOLOGY OF MICROBIAL BIOSYNTHESIS

正常和健康的细胞活动依赖于协调一致的相互作用 基因调控和蛋白质功能的重要性。我们的主要兴趣和长期- 学期目标是了解遗传和生化因素， 重要代谢途径磷酸戊糖的功能 通路这是一个代谢方案所需的代谢 几乎所有的生物体。 加上两种独特的酶， 核酮糖二磷酸羧化酶/加氧酶（RubisCO）和 磷酸核酮糖激酶（phosphoribulokinase，ERK），这条途径在一个纯粹的生物合成中起作用。 模式，使生物体获得利用二氧化碳作为 唯一的碳源。在这些条件下， 途径，包括普遍存在的催化剂，如转酮酶（TK）和 果糖1，6-二磷酸酶（FBPase），在原核生物和 真核生物，作为生物合成酶的功能，在相反的方向， 从它们在体内的通常作用中。编码几乎所有酶的基因 已经分离出了这种途径，并发现与不同的 非硫紫色细菌中的染色体操纵子（cbb调节子） 球形红细菌此外，转录激活蛋白 和一个编码传感激酶的基因，已经被发现可以调节 cbb基因的表达。其他监管要素也已 发现和这项研究的大部分是针对阐明 减轻感觉传导途径的调节机制 控制基因表达和最终的生物合成代谢。以来 暴露在不同水平的碳和氧中， 基因表达，协调一致的努力将集中在有关这种外部 刺激调节级联。 该项目的第二个主要目标将涉及结构研究- 的功能关系，FBPase，和TK。 因为他们的基因 表达为高活性重组蛋白， 纯化后，可以使用定点诱变方法 结合已知的X射线结构模型， 这些蛋白质的功能。这些都是非常重要的 代谢酶;例如TK是硫胺素代谢所必需的， 所有细胞及其动力学和化学性质的改变导致 严重的病理状况，包括营养缺乏， 酒精中毒、韦尼克-科萨科夫脑病和阿尔茨海默病。 FBPase是胚胎发生所必需的酶，PRO是其中之一 还原性戊糖磷酸途径所特有的。每例病患的 这里开发的重组系统具有实质上 增加了这些酶的可用信息，从而提供了一种不寻常的 有机会将细胞代谢的控制与功能联系起来， 关键催化剂的结构。

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.