Biochemistry of Bacterial Acetone Metabolism

细菌丙酮代谢的生物化学

• 批准号: 9630081
• 负责人: Scott Ensign
• 金额: $ 30.35万
• 依托单位: Utah State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9630081&HistoricalAwards=false
• 关键词: Biochemistry; Bacterial; Acetone; Metabolism

9630081 Ensign The objective of this project is to investigate the metabolic pathway(s) of acetone metabolism by microorganisms and the biochemical properties of the enzyme(s) which initiate acetone degradation. Acetone is a toxic molecule which is a product of microbial fermentation and mammalian starvation. Acetone is also a proposed intermediate metabolite in the aerobic oxidation of propane and isopropanol by some bacteria; it is an important industrial commodity synthesized in large quantities by the chemical industry. Studies of aerobic and anaerobic acetone-degrading bacteria have provided evidence for the existence of two distinct routes for acetone degradation: an initial hydroxylation reaction producing acetol (hydroxyacetone), or an initial carboxylation reaction producing acetoacetate. However, no acetone-degrading enzyme has been purified or studied in vitro, and consequently, little is known about the properties of these enzymes or the mechanisms they employ for acetone conversions. Our studies are focused primarily on Xanthobacter strain Py2, an aerobic bacterium capable of utilizing acetone and other ketones as carbon and energy sources. Preliminary studies performed in our laboratory have demonstrated that acetone metabolism in Xanthobacter Py2 proceeds by a carboxylation reaction producing acetoacetate as the product. We have recently succeeded in reconstituting this carboxylation reaction in vitro. This represents the first demonstration of in vitro acetone carboxylation in a bacterial system. The specific objectives for this project period are to (1) purify to homogeneity and biochemically characterize the acetone carboxylase of Xanthobacter Py2; (2) kinetically and mechanistically characterize acetone carboxylase; (3) initiate the isolation, cloning, and characterization of gene(s) encoding acetone carboxylase; and (4) perform comparative studies of acetone metabolism in the propane-oxidizing bacterium Mycobacteriurn vaccae strain JOB5. Although the pathways and b iochemistry of microbial acetone formation have been studied in detail, there is a lack of information on the pathways and biochemistry of acetone degradation. The research to be conducted is intended to fill this gap in our understanding of biological acetone cycling and provide important fundamental insights into biochemical mechanisms of microbial acetone catabolism. %%% The objective of this project is to investigate metabolic pathways of acetone degradation by microorganisms and the biochemical properties of the enzymes which are required for acetone degradation. Acetone is an important industrial solvent, and is one of many organic compounds of environmental concern due to its toxicity. In addition to its production in industry, acetone is also formed by some microorganisms as a product of fermentation. Acetone is also formed by mammals, including humans, during starvation. In diabetics, acetone is formed as a product of incomplete carbohydrate breakdown, and accumulates to high levels in the blood and urine. The metabolism (breakdown) of acetone has been observed in both mammals and microorganisms, although the details and significance of these processes are not fully understood. This project is an investigation of the metabolism of acetone by bacteria that are able to grow at the expense of acetone as a source of carbon and energy. Our goals are to determine the sequence of reactions involved in acetone degradation and to determine how acetone is degraded at the molecular level. These studies will provide important new information on biological strategies for the degradation of toxic organic compounds. ***

9630081 Ensign本项目的目的是研究微生物丙酮代谢的代谢途径和启动丙酮降解的酶的生化特性。 丙酮是一种有毒分子，是微生物发酵和哺乳动物饥饿的产物。 丙酮也是一些细菌在丙烷和异丙醇的有氧氧化中提出的中间代谢物;它是化学工业大量合成的重要工业商品。对好氧和厌氧丙酮降解细菌的研究提供了证据，证明丙酮降解存在两种不同的途径：产生丙酮醇（羟基丙酮）的初始羟基化反应，或产生乙酰乙酸的初始羧化反应。 然而，没有丙酮降解酶已被纯化或在体外研究，因此，很少有人知道这些酶的性质或它们用于丙酮转化的机制。 我们的研究主要集中在黄原胶菌株Py 2上，这是一种能够利用丙酮和其他酮作为碳源和能源的好氧细菌。 在我们的实验室进行的初步研究表明，丙酮代谢在黄嘌呤Py 2进行的羧化反应，产生乙酰乙酸作为产品。 我们最近成功地在体外重建了这种羧化反应。 这代表了在体外丙酮羧化在细菌系统中的第一个演示。 本项目期间的具体目标是：（1）纯化至均一并对黄原胶Py 2的丙酮羧化酶进行生化表征;（2）对丙酮羧化酶进行动力学和机械学表征;（3）开始分离、克隆和表征编码丙酮羧化酶的基因;和（4）在丙烷氧化细菌母牛分枝杆菌菌株JOB 5中进行丙酮代谢的比较研究。 虽然微生物丙酮生成的途径和B生物化学已被详细研究，但丙酮降解的途径和生物化学信息缺乏。 要进行的研究是为了填补这一空白，我们的理解生物丙酮循环，并提供重要的基本见解微生物丙酮催化剂的生化机制。 %%% 本项目的目的是研究微生物降解丙酮的代谢途径和丙酮降解所需酶的生化性质。 丙酮是一种重要的工业溶剂，并且由于其毒性而成为许多环境关注的有机化合物之一。 除了在工业上生产外，丙酮也是由一些微生物发酵形成的产物。 丙酮也由哺乳动物，包括人类，在饥饿期间形成。在糖尿病患者中，丙酮是碳水化合物不完全分解的产物，并在血液和尿液中积累到高水平。 在哺乳动物和微生物中都观察到丙酮的代谢（分解），尽管这些过程的细节和意义尚未完全了解。 该项目是一个研究丙酮代谢的细菌，能够生长在丙酮作为碳和能源的代价。 我们的目标是确定丙酮降解的反应顺序，并确定丙酮是如何在分子水平上降解的。 这些研究将为降解有毒有机化合物的生物学策略提供重要的新信息。 ***