Collaborative Research: Simplifying metabolic pathways by wiring redox proteins together

PI：Koder，罗纳德/ Banta，Scott A.提案编号：1403748 /1402913机构：CUNY City College /哥伦比亚大学标题：合作研究：通过将氧化还原蛋白连接在一起来简化代谢途径酶蛋白之间的能量转移通常使用小分子辅因子，如NAD（H）和NADP（H）来完成。 该系统在生物系统中是有利的;然而，在技术应用中，由于包括它们的高成本和低稳定性在内的几个原因，这些辅因子的使用是不期望的。 蛋白质设计和蛋白质工程的进步使合成生物学取得了巨大的进步，可以创造和表征具有新的非天然功能的新蛋白质。 在这个项目中，一种新的蛋白质将通过将两种辅因子依赖性酶偶联在一起来制备。 为了消除对辅因子的需要，酶将与设计的“钉”肽系统连接，该系统包含氧化还原中心，使酶之间的电子转移成为可能。 这将把蛋白质“连接”在一起，这样它们都不需要不稳定的辅因子分子来进行活性。 结合起来，这种新的酶复合物将具有自然界中没有的活性。 这将为将其他氧化还原蛋白连接在一起，以实现在未来合成生物学项目中消除辅因子需求的目标指明一条前进的道路。 该小组由来自纽约城市学院和哥伦比亚大学的PI组成。 这个合作项目的总体目标是利用蛋白质工程将两个不相关的氧化还原蛋白连接在一起，创造一种新的无辅因子的酶促反应。 研究人员将试图证明两种氧化还原蛋白连接在一起形成乒乓氧化还原酶的第一个例子，该酶具有自然界中未发现的活性。 他们将从两种已知的氧化还原酶开始：甲酸脱氢酶和乳酸脱氢酶。 这些酶将通过一种新的“胶原蛋白钉”系统连接起来，该系统将实现特定的自组装和引入氧化还原中心，用于两个酶活性位点之间的直接电子转移。 最终的酶将具有全新的功能：丙酮酸还原为乳酸，同时甲酸氧化为二氧化碳。 新的动力学机制和所得生物催化剂的活性将被广泛表征。 然后，这种方法可以扩展到结合联合收割机额外的酶，创造新的无辅因子的生物催化剂，用于工业上重要的氧化和还原反应。这种方法将允许将具有连续动力学机制的联合收割机氧化还原酶组合成具有合成氧化还原中心的单个乒乓酶。 该研究与教育活动相结合，向本科生，研究生和纽约市公立高中教师介绍生物物理学的跨学科科学。 这些努力将鼓励年轻学生追求科学，技术，工程和数学的职业生涯。CBET部门的生物技术，生物化学和生物质工程项目的这个奖项是由分子和细胞生物学部门的系统和合成生物学项目共同资助的。

PI's: Koder, Ronald / Banta, Scott A.Proposal Numbers: 1403748 / 1402913Institutions: CUNY City College / Columbia UniversityTitle: Collaborative Research: Simplifying metabolic pathways by wiring redox proteins together Energy transfer between enzymatic proteins is often accomplished using small molecule cofactors, such as NAD(H) and NADP(H). This system is advantageous in biological systems; however, in technology applications the use of these cofactors is undesirable for several reasons including their high cost and low stability. Advances in protein design and protein engineering have enabled tremendous advances in synthetic biology where new proteins with novel, unnatural functions can be created and characterized. In this project a new protein will be made by coupling two cofactor-dependent enzymes together. In order to eliminate the need for the cofactor, the enzymes will be connected with a designed 'staple' peptide system that contains redox center that enable electron transfer between the enzymes. This will 'wire' the proteins together so that neither one requires the unstable cofactor molecules for activity. Combined, this new enzyme complex will have an activity that is not found in nature. And this will chart a path forward for wiring other redox proteins together towards a goal of eliminating cofactor requirements in future synthetic biology projects. The team consists of PIs from City College of New York and Columbia University. There will be several outreach activities associated with this project including interactions with local high school teachers and students.The overall goal of this collaborative project is to use protein engineering to wire two unrelated redox proteins together creating a novel cofactor-less enzymatic reaction. The investigators will attempt to demonstrate the first example of the wiring of two redox proteins together to form a ping pong redox enzyme with an activity not found in nature. They will start with two known redox enzymes: formate dehydrogenase and lactate dehydrogenase. These enzymes will be connected by a novel 'collagen staple' system that will enable specific self-assembly and the introduction of redox centers for direct electron transfer between the two enzyme active sites. The final enzyme will have a completely novel function: pyruvate reduction to lactate with the concomitant oxidation of formate to carbon dioxide. The new kinetic mechanism and activity of the resultant biocatalyst will be extensively characterized. This approach can then be extended to combine additional enzymes, creating new cofactor-less biocatalysts for industrially important oxidation and reduction reactions. This approach will allow to combine redox enzymes with sequential kinetic mechanisms into a single ping pong enzyme with a synthetic redox center. The research is integrated with educational activities which introduces undergraduates, graduate students and New York City Public High School teachers to the interdisciplinary science of biophysics. These efforts will encourage young students to pursue careers in Science, Technology, Engineering and Mathematics.This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.