Structural Biology of Complex Enzymes

项目摘要 天然产物的生物合成途径为稀有的化学基团提供了巨大的合成潜力 在生物学上。这些酶从初级代谢适应到这些次级代谢中的新功能， 代谢物途径是许多应用的生物催化潜力的主要来源。本工程 结构和功能的几种酶，催化不寻常的反应，在生物合成的天然 产品将被调查。蛋白质晶体结构将为功能测定的发展提供信息， 这将被用来测试基于结构的假设，机制，底物选择性和产品 的特异性这些酶来自细菌和真菌的生物合成途径，用于几种天然产物 类，包括聚酮，非核糖体肽和azaphilones。这四种酶系统具有 生物催化剂在其天然途径内或外的巨大发展潜力， 理解蛋白质在自然界中的进化，扩大酶化学的范围， 工程新功能在第一个系统中，细菌酶具有独特的酰基转移， 脱羧作用在两种生物合成途径中起作用。这一双重功能具有广泛的代表性 更大的超家族的独立分支;它们如何在一个酶中组合将被确定。 在第二个系统中，与氧化还原酶相关的酶家族在卸载酰胺中起作用 非核糖体肽合成酶的载体蛋白的产物。在第三种制度中，一个大家庭 的真菌黄素依赖性单加氧酶催化芳族底物的羟基化反应， azaphilone和相关途径。选择的位点和立体特异性的结构基础 将确定家庭成员，以开发一个生物催化剂小组， 脱芳烃反应在第四个系统中，宏观周期形成的决定因素和 将研究五种酶的扩大底物范围的可能性。

Project Summary Natural product biosynthetic pathways harbor a vast synthetic potential for chemical groups that are rare in biology. Enzymes that were adapted from primary metabolism to novel functions in these secondary- metabolite pathways are a major source biocatalytic potential for many applications. In this project, the structure and function of several enzymes that catalyze unusual reactions in the biosynthesis of natural products will be investigated. Protein crystal structures will inform the development of functional assays, which will be used to test structure-based hypotheses about mechanism, substrate selectivity and product specificity. The enzymes are from bacterial and fungal biosynthetic pathways for several natural product classes, including polyketides, nonribosomal peptides and azaphilones. The four enzyme systems have great potential for biocatalyst development within or outside their natural pathways, for advancing our understanding of protein evolution in nature, for expanding the scope of enzyme chemistry, and for engineering new function. In the first system, a bacterial enzyme has distinct acyl transfer and decarboxylation functions in two biosynthetic pathways. The dual functions are represented on widely separated branches of the larger superfamily; how they are combined in one enzyme will be determined. In the second system, an enzyme family related to oxidoreductases functions in offloading an amide product from the carrier protein of a nonribosomal peptide synthetase. In the third system, a large family of fungal flavin-dependent monooxygenases catalyzes hydroxylation reactions on aromatic substrates in azaphilone and related pathways. The structural basis for the site- and stereo-specificity of selected family members will be determined in order to develop a panel of biocatalysts for challenging dearomatization reactions. In the fourth system, the determinants of macrocycle formation and the possibilities for an expanded substrate scope of five enzymes will be investigated.