Enzymes of the Meta Fission Pathway

微生物生物合成和分解代谢途径的研究提供了关于酶机制的重要见解， 特异性、结构、调节和进化。利用这些见解和单个酶， 从合成新的药物化合物到生物修复的许多实际用途。此应用程序将 解决两个问题：抗生素抗肿瘤剂托马霉素的C环是如何形成的， 多环芳烃（PAH）通过一种主要的细菌途径降解。这两个问题都出现了 在对4-巴豆酸互变异构酶（4-OT）等互变异构酶超家族的研究过程中， 成员4-OT催化用于降解的裂变途径中的质子转移反应， 芳香烃. TomN是一个新发现的4-OT同源物，它引导我们找到了tomaymycin途径 因为据报道它使用非常不同的底物催化质子转移反应。第二个问题 出现于我们广泛使用乙炔化合物作为4-OT的机制探针和抑制剂， 其他超家族成员第一个问题的答案推进了我们对生物合成的理解 吡咯并[1，4]苯并二氮杂卓类化合物是一类重要的天然产物， 抗微生物和抗癌药物的来源以及药物先导。第二个问题的答案 我们对PAH降解途径的理解，特别是用于降解高分子的途径， 分子量大的多环芳烃，沿着分子量小的多环芳烃，毒性大，持久性强 环境污染物。综合运用机械学、生物化学、结构学和动力学 技术，这些问题将在三个具体目标解决。这些目标是：（1）确定 将L-多巴转化为托马霉素C环的反应;（2）鉴定TomK的关键元件 机制;和（3）建立机制和水合酶/醛缩酶的特异性的决定因素， 萘、菲和荧蒽降解途径。结果也将解决我们长期- 长期目标，这是为了更全面地了解结构和 在酶催化反应中起作用，并描述控制蛋白质结构、底物 识别和反应特异性。

Studies of microbial biosynthetic and catabolic pathways provide major insights about enzyme mechanism, specificity, structure, regulation, and evolution. Harnessing these insights and the individual enzymes has many practical uses ranging from the synthesis of new drug compounds to bioremediation. This application will address two questions: how the C ring of the antibiotic antitumor agent tomaymycin is made and how polycyclic aromatic hydrocarbons (PAHs) are degraded by one major bacterial pathway. Both questions arose in the course of our studies on 4-oxalocrotonate tautomerase (4-OT) and other tautomerase superfamily members. 4-OT catalyzes a proton transfer reaction in the meta-fission pathway for the degradation of aromatic hydrocarbons. TomN, a newly discovered 4-OT homologue, led us to the tomaymycin pathway because it reportedly catalyzes a proton transfer reaction using a very different substrate. The second question emerged from our extensive use of acetylene compounds as mechanistic probes and inhibitors of 4-OT and other superfamily members. The answer to the first question advances our understanding of the biosynthesis of a large and highly diverse group of natural products, the pyrrolo[1,4]benzodiazepines that are important sources of antimicrobial and anticancer drugs and drug leads. The answer to the second question advances our understanding of PAH degradative pathways, notably ones used for the degradation of higher molecular weight PAHs, which, along with the lower molecular weight PAHs, are highly toxic and persistent environmental contaminants. Using a combination of mechanistic, biochemical, structural, and kinetic techniques, these questions will be addressed in three specific aims. These aims are to: (1) identify the reactions that convert L-dopa to the C-ring of tomaymycin; (2) identify the key elements of the TomK mechanism; and (3) establish the mechanism and determinants of specificity for the hydratase/aldolases in the naphthalene, phenanthrene, and fluoranthene degradative pathways. The results will also address our long- term goals, which are to obtain a more comprehensive understanding of the relationship between structure and function in enzyme-catalyzed reactions and to delineate the factors that govern protein structure, substrate recognition, and reaction specificity.