Structure and Mechanisms of Flavin Monooxygenases

黄素单加氧酶的结构和机制

• 批准号: 6422377
• 负责人: David P Ballou
• 金额: $ 38.11万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6422377
• 关键词: Escherichia coli; X ray crystallography; chemical kinetics; cis trans isomerization; crystallization; enzyme mechanism; enzyme model; enzyme substrate; flavins; flavoproteins; intermolecular interaction; isozymes; model design /development; molecular dynamics; molecular genetics; molecular site; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; oxygenases; physical model; protein engineering; protein purification; protein structure function; site directed mutagenesis; spectrometry; time resolved data

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.

描述：(申请者提供)这项资助的研究将集中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 从分子的角度理解不同的家族是如何 黄素蛋白单加氧酶都可以通过形成共同的 氧中间体，C4a-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Flavin 提供灵敏的内置光谱报告器组，用于监控 反应的各个阶段，非常详细地检查了 包括蛋白质动态事件在内的个别反应是可能的。它是 计划详细研究四类此类酶：1)芳香族 羟基酶，似乎利用黄素氢过氧化氢作为一种 亲核试剂；2)环己酮单加氧酶，一种结合氧的酶 原子通过Baeyer-Villiger反应进入其底物，使用黄素C4a 作为亲核剂的过氧阴离子；3)广泛的底物耐受性酶 氧化多种含氮和含硫化合物；4)不同寻常 来自细菌来源的2-蛋白质组成的酶，执行 对羟基苯乙酸酯的羟基化。这些酶分布广泛。 在自然界中，并在动物的异种代谢中发挥重要作用， 土壤细菌对植物激素的生物合成及其生物修复过程。 它们还提供了合成手性化合物的可能性，这可能是 在药物开发中很有用。将广泛使用现场定向的 突变，基于已有的结构信息 对羟基苯甲酸羟基酶和苯酚羟基酶的测定 环己酮单加氧酶的结构也进行了规划。他们的反应 每种酶的作用机制将通过快速反应技术进行探索， 包括停流荧光和吸收光谱，通过核磁共振 同位素标记蛋白质，并通过固体核磁共振技术。广泛使用 也将在酶中引入人造黄素来代替 天然黄素作为活性中心结构和作用机理的探针剂。