Hydrolase Engineering by Circular Permutation

[0730312] lutz， Stefan生物催化在学术和工业研究中是一个快速发展的领域，用于在温和的反应条件下进行分子的有效对映和区域选择性转化。这些发展得到了酶工程和定向进化中新的革命性方法的支持，这些强大的工具使研究人员能够根据其底物和环境要求定制自然催化剂。a/B水解酶家族成员是功能最广泛和研究最广泛的生物催化剂之一。我们的实验室最近证明了一个家族成员，来自南极念珠菌（CALB）的脂肪酶B的催化性能可以通过使用称为循环排列（CP）的非常规蛋白质工程方法显着增强。然而，这种工程方法的结构和功能后果的预测框架是非常有限的。本研究将解决CP的两个基本方面。首先，我们将研究环状排列对蛋白质结构的影响，特别关注新的末端区域。利用我们现有的排列calb，我们将应用x射线晶体学、光谱技术和蛋白质工程来阐明工程脂肪酶N和c端的局部构象偏好，并评估它们与酶功能的相关性。其次，我们假设CP是a/ b水解酶折叠成员工程的通用方法，因为该家族的模块化设计，将催化残基置于结构保守的蛋白质核心，而底物结合由可互换的帽结构域决定。我们将在放射性农杆菌（EchA）环氧化物水解酶（制备不对称二醇的重要生物催化剂）上测试CP对其他a/B水解酶家族成员的改进的更广泛适用性。该项目将对教育基础设施产生影响，为未来各级科学家（本科生、研究生和博士后）提供优秀的培训和指导机会，包括女性和代表性不足的少数民族。位于化学和生物学的界面，概述的工作需要分子生物学，生物化学，物理化学和有机合成化学等领域的跨学科合作。研究小组的每个成员都将研究一个独立但相关的问题，灌输主人主人感，但也鼓励和要求定期沟通，这一过程将作为实验室日常互动的非正式部分，以及在每周小组会议上的正式报告中提供便利。研究小组的成员也被鼓励积极参加基础与应用进化中心（FAME）的月度会议和研讨会（并发表演讲）。FAME代表了亚特兰大范围内的12个pi和他们的学生。来自埃默里大学（Emory University）、莫尔豪斯学院（Morehouse College）和佐治亚理工学院（Georgia Institute of Technology）的化学、生物学、生物化学和化学工程系，他们对生物催化、定向分子进化和组合化学有着共同的兴趣。最后，这个研究项目的结果将通过学生在地方和国家科学会议上的报告来传播。参加这些活动还将把他们介绍给更广泛的科学界和新的研究领域。

0730312Lutz, Stefan Biocatalysis in academic and industrial research is a rapidly growing field for the efficient enantio and regioselective conversion of molecules under mild reaction conditions. These developments are supported by new revolutionary methods in enzyme engineering and directed evolution, powerful tools that enable researchers to customize Nature's catalysts to their substrates and environmental requirements. Among the functionally most versatile and intensely studied biocatalysts are members of the a/B hydrolase-fold family. Our laboratory recently demonstrated that the catalytic performance of one family member, the lipase B from Candida antarctica (CALB), can be significantly enhanced by using an unconventional protein engineering approach called circular permutation (CP). However, the predictive framework for the structural and functional consequences of this engineering method is very limited. This research will address two fundamental aspects of CP. Firstly, we will investigate the impact of circular permutation on protein structure with a particular focus on the new termini regions. Working with our existing permuted CALBs, we will apply x-ray crystallography, spectroscopic techniques and protein engineering to elucidate the local conformational preferences in the N and C-terminus of engineered lipases and assess their relevance to enzyme function. Secondly, we hypothesize that CP is a general method for engineering members of the a/B-hydrolase fold, due to the family's modular design, which places the catalytic residues in the structurally conserved protein core while substrate binding is determined by an interchangeable cap domain. We will test CP's broader applicability for the improvement of other a/B hydrolase-fold family members on the epoxide hydrolase from Agrobacterium radiobacter (EchA), an important biocatalyst for the preparation of asymmetric diols.The project will have an impact on the educational infrastructure by providing an excellent training and mentoring opportunity for future scientists on all levels (undergraduate, graduate, and postdoctoral), including women and underrepresented minorities. Situated at the interface of chemistry and biology, the outlined work requires interdisciplinary collaborations between the fields of molecular biology, biochemistry, physical chemistry and organic synthetic chemistry. Each members of the research team will work on an independent yet related problem, instilling the sense of ownership but also encouraging and requiring regular communication, a process that will be facilitate informally as part of the daily interactions in the laboratory and more formally in presentations during weekly group meetings. Members of the research group are also encouraged to actively participate (and give presentations) in the monthly meetings and seminars of the Center for Fundamental and Applied Evolution (FAME). FAME represents an Atlanta-wide group of 12 PIs and their students (approx. 90 people) from the chemistry, biology, biochemistry, and chemical engineering departments at Emory University, Morehouse College, and the Georgia Institute of Technology with a common interest in biocatalysis, directed molecular evolution and combinatorial chemistry. Finally, the results for this research project will be disseminated through student presentations at local and national scientific meetings. Attendance of these events will also introduce them to the broader scientific community and new research areas.