Hydrolase Engineering by Circular Permutation

通过循环排列进行水解酶工程

• 批准号: 0730312
• 负责人: Stefan Lutz
• 金额: $ 30万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0730312&HistoricalAwards=false
• 关键词: Hydrolase; Engineering; Circular; Permutation

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.

0730312Lutz，Stefan在学术和工业研究中的生物催化是在温和的反应条件下分子的高效对映体和区域选择性转化的一个快速增长的领域。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他们的底物和环境要求定制自然的催化剂。在功能最广泛和研究最深入的生物催化剂中，A/B水解酶折叠家族的成员。我们的实验室最近证明了一个家族成员，来自南极假丝酵母的脂肪酶B(Calb)，可以通过一种称为循环置换(CP)的非传统蛋白质工程方法来显著提高催化性能。然而，对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，特别是新的末端区域。与我们现有的置换CALB合作，我们将应用X射线结晶学、光谱技术和蛋白质工程来阐明工程脂肪酶N端和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是工程a/B-水解酶折叠成员的一种通用方法，因为该家族的模块化设计，将催化残基放置在结构保守的蛋白质核心中，而底物结合由一个可互换的帽结构域决定。我们将测试CP在改进其他A/B水解酶折叠家族成员方面的更广泛的适用性，该家族成员来自辐射农杆菌(ECHA)的环氧化物水解酶(ECHA)是制备不对称二元醇的重要生物催化剂。该项目将通过为未来各级(本科生、研究生和博士后)的科学家(包括女性和代表性不足的少数族裔)提供一个极好的培训和指导机会，对教育基础设施产生影响。这项工作位于化学和生物学的交界处，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致力于一个独立但又相关的问题，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得到非正式促进，并在每周小组会议期间的陈述中得到更正式的促进。还鼓励研究小组成员积极参加基础和应用进化中心(FAME)的每月会议和研讨会(并作报告)。FAME代表亚特兰大范围内由12名PI及其学生组成的团体(约90人)，来自埃默里大学、莫尔豪斯学院和佐治亚理工学院的化学、生物、生物化学和化学工程系，对生物催化、指导分子进化和组合化学有共同的兴趣。最后，这项研究项目的成果将通过学生在地方和国家科学会议上的陈述来传播。出席这些活动还将把它们介绍给更广泛的科学界和新的研究领域。