MOLECULAR DIVERSITY OF BACTERIAL BUTANE MONOOXYGENASES

描述：烷烃单加氧酶启动烷烃的氧化为 细菌的生长支持底物。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烷烃羟基酶。建议数 研究将集中在以丁烷单加氧酶为代表的C2-C5 烷烃利用率。关于丁烷的不寻常的多样性水平 三种细菌(布氏假单胞菌、CF8菌株和 在丁烷上生长的(母牛分枝杆菌)将被描述。知识 一般情况下，单加氧酶的活性将得到加强，结果可能导致 研究健康和疾病中的单加氧酶的新模型系统。一个 工作假说是丁烷单加氧酶 含有P450辅基，分离株CF8的丁烷单加氧酶 含有铜，而母牛分枝杆菌的丁烷单加氧酶含有二铁 中间。该提案的具体目标是：1.描述 完整细胞中丁烷氧化在生理水平上的多样性。 烷烃底物范围、产物、动力学常数、失活剂分布、 并将研究与烷烃氧化相关的蛋白质模式。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 目标细菌。纯化产物的物理性质和催化性能 将对蛋白质进行检查。3.相关基因的特征 丁烷氧化。编码每个丁烷单加氧酶的基因将 进行分离和测序，以与已知的单加氧酶进行比较，另外 基因(例如，还原酶成分)也将被分离。这个 将对基因的组织进行检查。基因表达实验 将会被执行。我们的长远计划是继续发展 丁烷氧化知识。感兴趣的主题超出了本主题的范围 建议包括丁烷中其他酶的特性 氧化途径，其他丁烷氧化菌的检查， 对编码有能力的基因的调控的研究 利用丁烷，以及详细的物理(光谱和结构)和 纯化的酶的动力学特性。

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.