Structure, Function, and Genetics of the E. Coli Phosphotriesterase Homolog

酶是将一种生物物质转化为另一种生物物质的天然蛋白质分子。酶通常具有非凡的选择性，因为它们能够从大量其他分子中识别并转化特定的底物分子。由于酶在生物体中是由基因编码的，其识别和转化底物的化学性质在进化过程中得到了优化和完善。该研究项目旨在研究土壤细菌中一种有趣的酶的进化优化，这种酶可以介导一种被称为磷酸三酯的农药和化学战剂的破坏。这种被称为磷酸三酯酶的酶在完成这项任务时效率很高，这是不寻常的，因为磷酸三酯的底物是大约40年前首次制备的合成化学物质。这表明优化这种酶的进化过程以前所未有的速度进行。我们已经从一种相关的细菌——大肠杆菌中发现了一种酶，它在结构上与磷酸三酯酶高度相似，我们认为大肠杆菌的同源物代表了磷酸三酯酶产生的一个新酶家族的成员。因此，我们有了一对进化相关酶的起点和终点，我们将开展旨在为这一进化过程提供有用的结构-功能线索的实验。托马斯·斯坎伦。研究计划本提案的研究计划侧重于从大肠杆菌中新发现的开放阅读框（ORF），这似乎是一种新酶快速进化的起点。土壤细菌的磷酸三酯酶能高效催化合成磷三酯杀虫剂和化学战剂的水解裂解。酶介导的裂解反应发生在扩散控制的极限，表明磷酸三酯酶在催化优化方面已经达到了进化的终点。考虑到它的磷酸三酯底物是在20世纪40年代和50年代首次合成的，因此为最大催化能力的进化提供了50年的时间窗口，这种酶已经达到进化完美的建议是令人惊讶的。我们最近在大肠杆菌中发现了一个新鉴定的染色体ORF与磷酸三酯酶具有很高的序列相似性，并且许多重要的活性位点残基在这两种酶之间是保守的。因此，我们将此命名为磷酸三酯酶同源蛋白（PHP）的大肠杆菌ORF似乎是磷酸三酯酶进化亚家族的成员，并为分子进化和蛋白质工程的有趣研究项目提供了基础，该项目可能在生产新的有机磷酸盐解毒剂方面具有实用价值。本研究计划描述了我们的计划，以表征PHP的生物物理和酶性质，并阐明该酶在大肠杆菌中发挥的自然功能。此外，我们计划以结构为导向的诱变研究，旨在以PHP为起点，追溯磷酸三酯酶的进化路径，以达到催化完美。最后，描述了实验室进化实验，目的是利用自然选择原理开发新的有机磷解毒酶。教学计划本提案的教学计划抓住了在加州大学旧金山分校创建一个强大的生物有机化学研究生教育项目的机会。讨论了几个重要的策略，包括开发新的以化学为基础的研究生课程的计划，将UCSF各个以生物为中心的部门的学生整合到这些课程中的方法，以及向少数民族本科生介绍化学/生物界面研究机会的计划。讨论了获奖期间预期的教学职责，并介绍了以往在茶经方面的成就和获奖情况。NSF表格1358 (1/94

Enzymes are natural protein molecules that convert one biological substance into another. Enzymes often have extraordinary selectivity properties in that they are able to recognize and convert a specific substrate molecule from a sea of other molecules. Because enzymes are encoded by genes in living organisms, their chemical properties of substrate recognition and conversion are optimized and refined by evolution. This research project is designed to study the evolutionary optimization of an interesting enzyme from soil bacteria that mediates the destruction of a family of pesticides and chemical warfare agents called phosphotriesters. The enzyme, called Phosphotriesterase, is highly efficient at carrying out this task and this is unusual because it's phosphotriester substrates are synthetic chemicals that were first prepared about forty years ago. This suggests that the evolutionary process for optimizing this enzyme happened at an unprecedented rate. We have discovered an enzyme from a related bacteria, E. coli, that is highly similar in structure to the Phosphotriesterase enzyme, and we believe that the E. coli homolog represents a member of a new family of enzymes from which Phosphotriesterase arose. Thus, we have the starting point and ending point for a pair of evolutionarily related enzymes, and we will carry out experiments designed to provide useful structure-function clues to this evolutionary process. Scanlan, Thomas S . Research Plan The research plan for this proposal focuses on a newly identified open reading frame (ORF) from Escherichia coli which appears to have been a starting point for rapid evolution of a new enzyme. The well-characterized enzyme Phosphotriesterase from soil bacteria catalyzes the hydrolytic cleavage of synthetic phosphotriester insecticides and chemical warfare agents in a hlghly efficient manner. The enzyme-mediated cleavage reaction occurs at the limit of diffusion control, suggesting that Phosphotriesterase has reached its evolutionary endpoint in catalytic optimization. The suggestion that this enzyme has achieved evolutionary perfection is amazing considering that its phosphotriester substrates were first synthesized in the 1940's and 50's, thus providing a fifty year time window for evolution of maximal catalytic power. We have recently discovered that a newly identified chromosomal ORF in E. coli has high sequence similarity to Phosphotriesterase, and many of the important active site residues are conserved between the two enzymes. This E. coli ORF, which we have named Phosphotriesterase Homolog Protein (PHP), therefore appears to be a member of the subfamily from which Phosphotriesterase evolved, and provides the basis for an interesting research project in molecular evolution and protein engineering that may have practical value in producing new organophosphate detoxification agents. This research proposal describes our plans to characterize the biophysical and enzymatic properties of PHP, and elucidate the natural function that this enzyme performs in E. coli. In addition, we plan structure-guided mutagenesis studies aimed at retracing Phosphotriesterase's evolutionary path to catalytic perfection using PHP as a starting point. Finally, laboratory evolution experiments are described, with the aim of developing new organophosphate detoxification enzymes using the principles of natural selection. Teachin~ Plan The teaching plan for this proposal seizes upon the opportunity of creating a strong graduate education program in bioorganic chemistry at UCSF. Several important strategies are discussed, including plans to develop new chemistry-based graduate courses, methods of integrating students from the various biology-centered departments at UCSF into these courses, and plans to introduce minority undergraduate students to research opportunities at the chemistry/biology interface. Teaching responsibilities anticipated during the award period are discussed, and previous accomplishments and awards in tea ching excellence are presented. NSF FORM 1358 (1/94) 2