Collaborative Research: Engineering a Multienzyme Complex with Synthetic and Mechanistic Goals

合作研究：设计具有合成和机械目标的多酶复合物

• 批准号: 1402675
• 负责人: Frank Jordan
• 金额: $ 32.1万
• 依托单位: Rutgers University Newark
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402675&HistoricalAwards=false
• 关键词: Collaborative; Research; Engineering; Multienzyme; Complex

Because life at the molecular level is the chemistry of carbon containing compounds, reactions that make carbon-carbon bonds are particularly valuable. A challenge for making compounds from carbon is the formation of closely related versions or isomers as products from a single reaction. The most closely related isomers are called stereoisomers, otherwise identical compounds, but mirror images of each other. The proposal envisions harnessing the power of biological transformations for carbon-carbon bond formation that make single stereoisomers for the production of practical compounds like pharmaceuticals. The reactions would not only be exquisitely specific, but also environmentally friendly, or "green" by using only natural materials and gentle conditions. The research plan involves the re-engineering of proteins using recombinant DNA technology so that they specifically make high-value products. The projects that make up the plan will enable interdisciplinary training of graduate students, postdoctoral fellows and undergraduates in the fields of enzymology, molecular biology, protein engineering, and chemical synthesis. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Frank Jordan and Edgardo T. Farinas, of Rutgers University-Newark Campus and the New Jersey Institute of Technology, respectively, to undertake enzymatic synthesis of chiral alpha-hydroxy ketones for pharmaceuticals, and fine chemicals for synthesis of larger molecules. For the first time the 2-oxoglutarate dehydrogenase multienzyme complex (OGHDc), both human and E. coli, and 2-hydroxy-3-oxoadipate synthase from Mycobacterium tuberculosis (HOAS), will be engineered to create these compounds. The principal investigators have already demonstrated that the first OGDHc component (E1o) is an efficient biocatalyst using the physiological substrate. To enhance the power of this carboligation reaction, they will: (1) Engineer residues from the substrate binding site selected from X-ray structures of the E1os and HOAS to accept a variety of substrates for efficient enzymatic synthesis of alpha-hydroxy ketones, with high enantiomeric excess (ee) and yield; (2) Explore alterations in the chemical nature of donor 2-oxo acid and of acceptor aldehyde. (3) Advance the field of biocatalysis by engineering not only E1os but the second E2o core component of OGDHc to accept unnatural substrates leading to acyl-coenzyme A analogues participating in many metabolic pathways; (4) Explore the mechanism of intra-E2o component acyl transfer to coenzyme A.

因为分子水平上的生命是含碳化合物的化学，所以形成碳-碳键的反应特别有价值。从碳制造化合物的一个挑战是形成密切相关的版本或异构体，作为单一反应的产物。关系最密切的异构体被称为立体异构体，否则是相同的化合物，但彼此镜像。该提案设想利用生物转化的力量来形成碳-碳键，这些转化产生单一的立体异构体，用于生产实际的化合物，如药物。这种反应不仅精致具体，而且环保，或者只使用天然材料和温和的条件，或者说是绿色的。该研究计划涉及使用重组DNA技术对蛋白质进行重新设计，以便它们专门制造高价值的产品。组成该计划的项目将使研究生、博士后研究员和本科生在酶学、分子生物学、蛋白质工程和化学合成领域进行跨学科培训。该奖项将资助罗格斯大学纽瓦克校区的Frank Jordan博士和新泽西理工学院的Edgardo T.Farinas博士进行制药用手性α-羟基酮的酶促合成，以及用于合成大分子的精细化学品的酶促合成。人类和大肠杆菌的2-氧戊二酸脱氢酶多酶复合体(OGHDc)和结核分枝杆菌的2-羟基-3-氧己二酸合成酶(HOAs)将首次被设计成产生这些化合物。主要研究人员已经证明了第一个OGDHc组分(E10)是一种使用生理底物的高效生物催化剂。为了加强这种碳化反应的能力，他们将：(1)从底物结合部位的残基中选择E1os和HOA的X射线结构来接受各种底物，以高效地酶促合成高对映体过量(Ee)和高产率的α-羟基酮；(2)探索给体2-氧基酸和受体醛的化学性质的变化。(3)通过改造E1os和OGDHc的第二个E2O核心成分以接受导致酰基辅酶A类似物参与多种代谢途径的非天然底物来推进生物催化领域；(4)探索E2O内组份酰基转移到辅酶A的机制。