MOLECULAR DIVERSITY OF BACTERIAL BUTANE MONOOXYGENASES

描述：烷烃单加氧酶启动烷烃的氧化， 支持细菌生长的基质。 氧化的单加氧酶 C2-C5烷烃没有得到很好的研究，与C1烷烃颗粒和 可溶性甲烷单加氧酶和C6-C12烷烃羟化酶。 拟议 研究将集中在丁烷单加氧酶作为C2-C5的代表 烷烃利用者。 丁烷的多样性达到了不同寻常的水平 在三种细菌（Pseudomonasbutanovora，分离物CF 8，和 将表征在丁烷上生长的母牛分枝杆菌。 知识 一般来说，单加氧酶的活性将得到增强，结果可能导致 用于研究健康和疾病中单加氧酶的新模型系统。 一 工作假设是P. butanovora的丁烷单加氧酶 含有P450辅基，分离物CF 8的丁烷单加氧酶 含有铜，和M的丁烷单加氧酶。母牛含有二铁 中心 该提案的具体目标是：1。 表征 完整细胞中生理水平丁烷氧化的多样性。 烷烃底物范围，产物，动力学常数，灭活剂概况， 和与烷烃氧化相关的蛋白质模式将被检查。 2. 丁烷单加氧酶的纯化及性质研究。 丁烷 单加氧酶和相关的还原酶组分将从该酶中纯化。 目标细菌。 纯化的催化剂的物理和催化性质 将检测蛋白质。 3. 相关基因的表征 丁烷氧化。 编码每种丁烷单加氧酶的基因将 分离并测序以与已知的单加氧酶进行比较， 还将分离基因（例如还原酶组分的基因）。 的 将检查基因的组织。 基因表达实验 将被执行。 我们的长远计划是继续发展 丁烷氧化的知识。 本文范围之外的感兴趣的主题 建议包括丁烷中其他酶的特性 氧化途径，检查其他丁烷氧化细菌， 检查编码以下能力的基因的调控控制 利用丁烷，和详细的物理（光谱和结构）， 纯化酶的动力学表征。

DESCRIPTION: Alkane monooxygenases initiate the oxidation of alkanes as growth-supporting substrates for bacteria. Monooxygenases which oxidize C2-C5 alkanes are not well studied, in contrast to C1 alkane particulate and soluble methane monooxygenases and C6-C12 alkane hydroxylase. The proposed research will focus on butane monooxygenases as representatives of C2-C5 alkane utilizers. An unusual level of diversity with regard to butane monooxygenases in three bacteria (Pseudomonas butanovora, isolate CF8, and Mycobacterium vaccae) that grow on butane will be characterized. Knowledge of monooxygenases in general will be enhanced and the results could lead to new model systems for the study of monooxygenases in health and disease. A working hypothesis is that the butane monooxygenases of P. butanovora contains a P450 prosthetic group, the butane monooxygenase of isolate CF8 contains copper, and the butane monooxygenase of M. vaccae contains a diiron center. The specific aims of the proposal are: 1. Characterization of diversity in butane oxidation at the physiological level in intact cells. Alkane substrate ranges, products, kinetic constants, inactivator profiles, and protein patterns associated with alkane oxidation will be examined. 2. Purification and characterization of butane monooxygenase. Butane monooxygenase and associated reductase components will be purified from the targeted bacteria. Physical and catalytic properties of the purified proteins will be examined. 3. Characterization of the genes associated with butane oxidation. The genes coding for each butane monooxygenase will be isolated and sequenced for comparison to known monooxygenases, Additional genes (e.g. for reductase components) will also be isolated. The organization of the genes will be examined. Gene expression experiments will be performed. Our long-range plans are to continue to develop the knowledge of butane oxidation. Topics of interest outside the scope of this proposal include characterization of additional enzymes in the butane oxidation pathway, examination of other butane oxidizing bacteria, examination of the control of regulation of genes encoding the ability to utilize butane, and detailed physical (spectroscopic and structural) and kinetic characterizations of the purified enzymes.