Stopped-flow spectrometer for analysis of reaction kinetics

项目摘要 人类癌细胞中的多胺生物合成是一个长期存在的治疗靶点。越来越多的证据 表明肠道微生物产生的多胺调节结肠癌和胰腺癌的进展。 肠道细菌生物合成多胺亚精胺的途径与人类途径不同 使得这种细菌途径成为独特的药物靶点。这一途径中的两种中心酶， 脱氢酶和羧胸苷脱羧酶，只得到有限的表征。我们提出 解析羧基胸苷脱氢酶的第一个结构，并建立动力学和结构模型 描述了羧基胸苷脱氢酶和羧基胸苷脱羧酶在 来自拟杆菌属和梭菌属的主要肠道成分。两种羧基胸苷脱氢酶 和脱羧酶使用具有独特的光谱“手柄”的辅酶，可以通过停止- 流动光谱法来辨别它们各自的催化循环的动力学步骤。一个彻底的结构和 对酶机制的功能理解是机制指导药物设计的必要前提。 这项工作将扩大我们对一个关键的肠道微生物代谢途径：多胺的基本理解 生物合成

PROJECT SUMMARY Polyamine biosynthesis in human cancer cells is a long-standing therapeutic target. Accumulating evidence suggests that polyamine production by gut microbes modulates the progression of colon and pancreatic cancer. Biosynthesis of the polyamine spermidine by gut bacteria follows a pathway distinct from the human pathway making this bacterial pathway a unique drug target. The two central enzymes in this pathway, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase, have received only limited characterization. We propose to solve the first structure of a carboxyspermidine dehydrogenase and to develop kinetic and structural models describing the function of both carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase in major gut constituents from the Bacteroides and Clostridium genera. Both carboxyspermidine dehydrogenase and decarboxylase use coenzymes with unique spectroscopic “handles” that can be interrogated by stopped- flow spectrometry to discern the kinetic steps of their respective catalytic cycles. A thorough structural and functional understanding of enzyme mechanisms is a necessary precursor to mechanism-guided drug design. This work will expand our fundamental understanding of a key gut microbe metabolic pathway: polyamine biosynthesis.