EAGER: Novel biocatalysts based on artificial metallo-cofactors in flavoproteins

Lutz(1546790)拟议的工作将产生关于重新设计黄素蛋白的活性部位--老黄酶(OYE)的概念验证数据，以进行碳氢化学，目的是生产难以用非生物方法合成的化学品。为此，该提案将重点放在通过将具有催化活性的有机金属络合物整合到酶内部的活性辅因子位置来创建新的人工生物催化剂。这样形成的新生物催化剂将被评估它们催化C-C键形成反应的潜力。此外，替代人工假体辅助因子的一般方法具有广泛适用于化学合成和化学转化为有用产品的许多领域的潜力。该项目还将以教育部分为特色，培训学生在生物工程领域取得成功所需的各种工程和科学学科，并将继续由首席研究员(PI)参与一些旨在提高大亚特兰大地区代表性不足的少数族裔对STEM领域的兴趣和教育的项目。该提议利用了黄素依赖的氧化还原酶作为能够在实验室规模上进行生物催化的酶的用途。具体地说，Pi和他的学生将通过将催化效率高的铜-双恶唑啉(铜-box)络合物整合到旧黄酶的活性位置来创建新的人工生物催化剂。尽管具有挑战性，但PI已经处于有利地位，可以成功地进行这种合成，这是基于他在Oye实验室三年多的工作，已经证明这种酶的天然氧化还原活性得到了实质性的改善。这些研究证实了PI在不损害酶的整体结构完整性的情况下对活性部位进行实质性改变的能力。然后，将通过苯乙烯和重氮乙酸乙酯的不对称环丙烷化反应来测试修饰后的酶的催化活性，重氮乙酸乙酯是形成C-C键的基准反应。通过探索黄素蛋白的新可能性，基于有机金属配体的人工辅助因子的加入可以极大地扩大生物催化的工具箱。

Lutz (1546790)The proposed work will generate proof-of-concept data on re-engineering the active site of the flavoprotein, Old Yellow Enzyme (OYE), to perform hydrocarbon chemistry aimed at producing chemicals that are difficult to synthesize by non-biological approaches. To this end, the proposal will focus on creating new artificial biocatalysts by integrating catalytically active organometallic complexes in the active cofactor site in the interior of the enzyme. The new biocatalysts so-formed will be assessed for their potential to catalyze C-C bond forming reactions. Moreover, the general approach of substituting artificial prosthetic co-factors has potential for general applicability to many areas of chemical synthesis and chemical conversion to useful products. The project will also feature educational components to train students in the diverse engineering and scientific disciplines required for success in the bioengineering area, and will continue the principal investigator's (PI's) involvement in a number of programs aimed at increasing interest and education in STEM areas amongst underrepresented minorities in the greater Atlanta area. The proposal capitalizes on the utility of flavin-dependent oxidoreductases as enzymes capable of biocatalysis at the laboratory scale. Specifically, the PI and his students will create new artificial biocatalysts by integrating catalytically efficient copper-bis(oxazoline)(Cu-BOX) complexes in the active site of Old Yellow Enzyme. Although challenging, the PI is well positioned to carry out this synthesis successfully based on more than three years of work in his lab on OYE which has already demonstrated substantial improvements in the enzyme's native redox activity. These studies have confirmed the PI's capability to make substantial changes in the active site without compromising the overall structural integrity of the enzyme. The modified enzymes will then be tested for catalytic activity via asymmetric cyclopropanation of styrene and ethyl diazoacetate, a benchmark reaction for C-C bond formation. By exploring new possibilities for the flavoprotein, the incorporation of artificial cofactors based on organo-metallic ligands could dramatically expand the toolbox for biocatalysis.