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

因为分子水平上的生命是含碳化合物的化学过程，所以形成碳-碳键的反应特别有价值。 从碳制造化合物的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。 最密切相关的异构体称为立体异构体，否则相同的化合物，但彼此镜像。 该提案设想利用生物转化的力量形成碳-碳键，使单一的立体异构体用于生产实际的化合物，如药物。 这些反应不仅是精细的，而且是环境友好的，或者说是“绿色”的，只使用天然材料和温和的条件。 该研究计划涉及使用重组DNA技术重新设计蛋白质，以便它们专门制造高价值产品。 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的跨学科培训成为可能。有了这个奖项，化学部的生命过程化学计划正在资助弗兰克·乔丹博士和埃德加多·T。法里纳斯，罗格斯大学纽瓦克校区和新泽西理工学院，分别进行酶法合成手性α-羟基酮的药物，和精细化学品的合成更大的分子。本文首次报道了人和大肠杆菌的2-酮戊二酸脱氢酶多酶复合物（OGHDc）。大肠杆菌的2-羟基-3-氧代己二酸合酶和来自结核分枝杆菌（HOAS）的2-羟基-3-氧代己二酸合酶将被工程化以产生这些化合物。主要研究人员已经证明，第一个OGDHc组分（E1 o）是使用生理底物的有效生物催化剂。为了增强这种碳配反应的能力，他们将：（1）从E1 os和HOAS的X射线结构中选择的底物结合位点设计残基，以接受各种底物，用于高效酶促合成α-羟基酮，具有高对映体过量（ee）和产率;（2）探索供体2-含氧酸和受体醛的化学性质的改变。(3)通过改造OGDHc的第二个E2 o核心组分，使其接受非天然底物，从而产生参与多种代谢途径的酰基辅酶A类似物，从而推进生物催化领域的发展;（4）探索E2 o组分内酰基转移到辅酶A的机制。

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.