Structure and Mechanisms of Flavin Monooxygenases

描述：（由申请人提供）本基金资助的研究将 着重于黄素蛋白单加氧酶的结构-功能关系， 生物化学、生物物理和蛋白质工程方法。一个主要目标是 从分子的角度来理解不同家族的 黄素蛋白单加氧酶都可以通过形成共同的 氧中间体，C4 a-过氧黄素，但仍然有非常不同的 底物特异性和调节机制。因为黄素 提供灵敏的内置光谱报告基团用于监测 反应的各个阶段，非常详细的检查， 包括蛋白质动力学事件的单独反应是可能的。是 计划详细研究四个主要类别的这种酶：1）芳香族 羟化酶，这似乎是功能使用黄素氢过氧化物作为一个 亲核试剂; 2）环己酮单加氧酶，一种掺入氧的酶 通过Baeyer-Villiger反应，使用黄素C4 a 过氧化物阴离子作为亲核试剂; 3）广泛的底物耐受酶，能够 氧化各种氮和硫化合物; 4）不寻常的 2-来自细菌来源的蛋白质组分酶，其执行 对羟基苯乙酸的羟基化。这些酶分布广泛 并在动物异生物质代谢中起重要作用， 植物激素生物合成和土壤细菌生物修复过程。 它们还提供了合成手性化合物的可能性， 用于药物开发。将广泛使用现场定向 诱变，基于结构信息已经可用于 对羟基苯甲酸羟化酶和苯酚羟化酶， 还设计了环己酮单加氧酶结构。反应 每种酶的作用机理将通过快速反应技术进行探索， 包括停流荧光和吸收光谱，通过NMR， 同位素标记的蛋白质，并通过固态NMR技术。广泛使用 也将由引入酶中的人工黄素代替 天然黄素作为活性部位结构和作用机制的探针。

DESCRIPTION: (provided by applicant) The research supported by this grant will focus on structure-function relationships of flavoprotein monooxygenases using biochemical, biophysical and protein engineering methods. A major goal is to develop an understanding in molecular terms of how the different families of flavoprotein monooxygenases can all function through the formation of common oxygen intermediates, the C4a-peroxyflavins, but nevertheless have very diverse substrate specificities and mechanisms of regulation. Because the flavin provides a sensitive built-in spectroscopic reporter group for monitoring individual stages of the reactions, a very detailed examination of the individual reactions, including protein dynamic events, is possible. It is planned to study in detail four main classes of such enzymes: 1) aromatic hydroxylases, which appear to function using the flavin hydroperoxide as a nucleophile; 2) cyclohexanone monooxygenase, an enzyme incorporating an oxygen atom into its substrate by a Baeyer-Villiger reaction, using the flavin C4a peroxide anion as a nucleophile; 3) broad substrate-tolerant enzymes capable of oxygenating a diverse array of nitrogen-and sulfur-compounds; 4) unusual 2-protein component enzymes from bacterial sources that carry out the hydroxylation of p-hydroxyphenylacetate. These enzymes are widely distributed in nature and play important roles in xenobiotic metabolism in animals, biosynthesis of plant hormones and bioremediation processes by soil bacteria. They also offer the possibility of synthesis of chiral compounds that could be useful in drug development. Extensive use will be made of site-directed mutagenesis, based on structural information already available for p-hydroxybenzoate hydroxylase and phenol hydroxylase, and determination of the structure of cyclohexanone monooxygenase is also planned. The reaction mechanism of each enzyme will be explored by rapid reaction techniques, including stopped-flow fluorescence and absorbance spectroscopy, by NMR of isotopic ally labeled protein, and by solid state NMR techniques. Extensive use will also be made of artificial flavins introduced into the enzymes in place of the natural flavins, as probes both of active site structure and mechanism.