The Core Metabolism of Rhodobacter sphaeroides

碳是所有已知生命形式的基本元素。几乎所有重要的细胞成分都含有碳化合物，在许多生物体中，这些含碳的生物分子同时被用来获取生长所需的能量。含碳生物分子被转化为更小的分子（前体代谢物），这些分子是生物体主要化学反应（代谢）的关键成分。这些前体代谢物可以作为其他细胞成分的基石，也可以在产生能量的化学反应中被分解。这些含碳生物分子的不同用途与微生物生长和能量需求所使用的碳源无关。因此，有必要了解前体代谢物的化学反应是如何被控制的，这样碳就可以流向用于构建细胞的分子。这一知识将使更有效地利用微生物产生有用的产品，包括那些具有生物技术重要性的产品（如生物燃料）。该项目将产生关于碳如何在生命系统中使用的基本新信息，并通过课堂和研究经验为下一代科学家，包括本科生和研究生，提供微生物生理学和遗传学方面的跨学科培训。对于每一种碳底物，必须控制前体代谢产物丙酮酸（C3）、磷酸烯醇丙酮酸（C3）和草酰乙酸（C4）的水平：这被称为C4/C3节点。重要的是，导致这些代谢物的途径及其命运取决于给定底物进入中心碳代谢的位置。在这个项目中，将研究参与C4/C3节点碳分割的酶的调节和活性，并解决为什么几种酶明显催化同一反应同时活跃的问题。球形红杆菌将用于扩大我们对碳同化的认识，因为在这种生物的无氧光异养生长过程中，能量代谢可能与碳同化分离。鉴于其在利用广泛的碳底物方面的通用性，圆木是本研究的理想选择。本研究将用于确定不同生长条件下C4-向c3前体代谢物转化所需的反应，以及丙酮酸和磷酸烯醇丙酮酸的相互转化所需的反应。利用球藻的遗传易感性，构建突变体进行比较实验，分析在特定的生长条件下需要哪些特定的酶。此外，重组蛋白的生化分析将确定动力学参数、潜在的异质组成、辅助因子特异性和C4/C3节点酶的翻译后调控。

Carbon is an essential element for all known forms of life. Nearly all important cellular constituents contain carbon compounds and in many organisms these carbon-containing biomolecules are simultaneously used to obtain energy for growth. Carbon-containing biomolecules are converted into smaller molecules (precursor metabolites) that are key components for the major chemical reactions (metabolism) of living organisms. These precursor metabolites can then serve either as the building blocks for other cellular constituents or can be broken down in chemical reactions that generate energy. These different uses of carbon-containing biomolecules occur regardless of the carbon sources that are used by microbes for their growth and energy requirements. Consequently, it is essential to understand the processes by which the chemical reactions of precursor metabolites are controlled so that carbon can flow towards the molecules that will be used to build cells. This knowledge will enable the more efficient use of microorganisms to generate useful products, including those (such as biofuels) of biotechnological importance. This project will generate fundamental new information on how carbon is used in living systems, and through classroom and research experiences will provide interdisciplinary training to the next generation of scientists, including undergraduate and graduate students, in microbial physiology and genetics. For every carbon substrate, the levels of the precursor metabolites pyruvate (C3), phosphoenolpyruvate (C3), and oxaloacetate (C4) have to be controlled: this is referred to as the C4/C3 node. Importantly, the pathways leading to these metabolites, as well as their fate, differ depending on where a given substrate has entered central carbon metabolism. In this project the regulation and activity of enzymes involved in the partition of carbon at the C4/C3 node will be examined and the question of why several enzymes apparently catalyzing the same reaction are active at the same time will be addressed. Rhodobacter sphaeroides will be used to expand our knowledge of assimilation of carbon since it is possible to disconnect energy metabolism from carbon assimilation during anoxygenic photoheterotrophic growth of this organism. R. sphaeroides is ideally suited for this study given its versatility in utilizing a wide spectrum of carbon substrates. This research will be used to determine the reactions required for 1) the conversion of C4- to C3-precursor metabolites under different growth conditions and 2) the inter-conversion of pyruvate and phosphoenolpyruvate. By capitalizing on the genetic tractability of R. sphaeroides mutants will be constructed for comparative experiments analyzing which specific enzymes are required under particular growth conditions. In addition, biochemical analyses of recombinant proteins will determine kinetic parameters, potential heteromeric composition, cofactor specificity, and posttranslational regulation of enzymes at the C4/C3 node.