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增长的领域，用于在温和的反应条件下有效地进行分子的对映体和区域选择性转化。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。在功能上最通用和深入研究的生物催化剂是a/B水解酶折叠家族的成员。我们的实验室最近证明，一个家庭成员，脂肪酶B从念珠菌anastritica（CALB）的催化性能，可以显着提高通过使用一种非传统的蛋白质工程方法称为循环排列（CP）。然而，这种工程方法的结构和功能后果的预测框架非常有限。这项研究将解决CP的两个基本方面。首先，我们将研究循环排列对蛋白质结构的影响，特别关注新的末端区域。与我们现有的排列CALB工作，我们将应用X射线晶体学，光谱技术和蛋白质工程，以阐明本地的构象偏好的N和C-末端的工程脂肪酶，并评估其相关性酶的功能。其次，我们假设CP是一种通用的方法，工程成员的a/B-水解酶折叠，由于家庭的模块化设计，将催化残基的结构保守的蛋白质核心，而底物结合是由一个可互换的帽结构域。我们将测试CP在改进放射性土壤杆菌（EchA）环氧化物水解酶上的其他a/B水解酶折叠家族成员的更广泛的适用性，EchA是制备不对称二醇的重要生物催化剂。该项目将通过为各级未来科学家提供极好的培训和指导机会，对教育基础设施产生影响（本科生，研究生和博士后），包括妇女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领域之间的跨学科合作。研究团队的每个成员都将致力于一个独立但相关的问题，灌输主人翁意识，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一部分非正式地促进，并在每周小组会议期间更正式地进行演示。研究小组的成员也被鼓励积极参与（并发表演讲）在基础和应用进化中心（FAME）的每月会议和研讨会。FAME代表了一个由12个PI和他们的学生组成的全亚特兰大集团（约100名学生）。来自埃默里大学、莫尔豪斯学院和格鲁吉亚理工学院的化学、生物学、生物化学和化学工程系的90人），他们对生物催化、定向分子进化和组合化学有共同的兴趣。最后，该研究项目的结果将通过学生在地方和国家科学会议上的演讲进行传播。参加这些活动还将向他们介绍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.