SusChem: New Methodologies in Biocatalysis

利用传统化学方法，生产和纯化高价值分子（香料、香精、染料、抗生素）往往消耗大量能源，产品收率低，并产生大量危险废物。该项目的目标是使用最小化浪费和最大化产量的策略来制造具有商业价值的分子，以便工业可以采用这些方法，从而改进化学过程。酶——自然界的蛋白质催化剂——将被用来代替传统的方法，因为它们是由可再生资源生产的，它们在水中而不是有机溶剂中工作，而且它们是完全可生物降解的。除了开发新的反应策略外，还将确定酶的三维结构。计算机建模将显示它们的形状如何变化，这将极大地影响它们的行为。这些知识将用于改进酶，使它们更适合化学合成的需要。如果取得成功，这些努力可能会产生一个更加可持续和环保的化学工业。PI积极参与佛罗里达大学的生命科学（S4L）计划，将直接有助于培养高素质的STEM劳动力。该项目的第一部分重点是修饰酶来催化硫代克拉森缩合和β -酮酸脱羧，以便在一个简单的单反应器系统中产生手性α -氨基- β -羟基酮。对映选择性官能团相互转换是第二部分的主题。这集中在一个酶家族（老黄酶，OYEs），立体选择性地减少缺乏电子的烯烃。该家族的成员具有高度保守的氨基酸序列和基本相同的x射线晶体结构，但表现出广泛不同的立体选择性，这似乎与催化循环过程中改变结构的蛋白质环有关。分子动力学计算表明，不同酶之间的环具有非常不同的动力学性质，这些动力学性质可能被分离到环中的单个氨基酸上。该位置的位点饱和诱变将产生突变体，这些突变体将通过实验（关于底物接受性和立体选择性）和计算（以确定动态特性的差异）进行检查。该回路中的其他位置将通过实验和计算来表征，以更好地定义这一重要结构特征如何控制底物结合。对结构动力学在酶活性调节中的作用的基本理解将极大地扩展设计具有所需功能的蛋白质的能力，为可持续的生物制造业做出贡献。

Using traditional chemistry, the production and purification of high value molecules (fragrances, flavors, dyes, antibiotics) often uses a lot of energy, has low yield of product, and generates significant quantities of hazardous wastes. The goal of this project is to make commercially important molecules using strategies that minimize waste and maximize yield, so that industry can employ these approaches and thereby improve chemical processes. Enzymes - Nature's protein catalysts - will be used instead of traditional methods, since they are produced from renewable resources, they work in water instead of organic solvents, and they are completely biodegradable. In addition to developing new reaction strategies, the three-dimensional structures of the enzymes will be determined. Computer modeling will indicate how their shapes change, which can dramatically impact their behavior. This knowledge will be put to use to improve the enzymes, making them better-suited to the needs of chemical synthesis. If successful, these efforts could result in a much more sustainable and environmentally-friendly chemical industry. The PI's active involvement with the Science for Life (S4L) program at the University of Florida will contribute directly to the development of a highly capable STEM workforce.The first part of this project focuses on modifying enzymes to catalyze thio-Claisen condensation and beta-keto acid decarboxylation, in order to create a chiral alpha-amino-beta-hydroxy ketone in a simple, single-reactor system. Enantioselective functional group interconversion is the subject of the second part. This focuses on a family of enzymes (Old Yellow Enzymes, OYEs) that stereoselectively reduce electron-deficient alkenes. Members of this family have highly conserved amino acid sequences and essentially identical x-ray crystal structures, yet exhibit widely varying stereoselectivities that appears to be related to a protein loop that changes structure during the catalytic cycle. Molecular dynamics calculations revealed that the loops have very different dynamic properties from enzyme to enzyme, which may be isolated to a single amino acid in the loop. Site-saturation mutagenesis at this position will generate mutants that will be examined both experimentally (regarding substrate acceptance and stereoselectivity) and computationally (to identify differences in dynamic properties). Additional positions in this loop will be characterized by both experiment and computation to better define how this important structural feature constrols substrate binding. A fundamental understanding of the role that structural dynamics plays in the regulation of enzyme activity would dramatically expand the ability to design proteins with desired functionality, contributing to a sustainable biomanufacturing industry.