SusChem: New Methodologies in Biocatalysis

SusChem：生物催化新方法

• 批准号: 1705918
• 负责人: Jon Stewart
• 金额: $ 60.57万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1705918&HistoricalAwards=false
• 关键词: SusChem; New; Methodologies; Biocatalysis

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.

使用传统化学，高价值分子（香料，香料，染料，抗生素）的生产和纯化通常使用大量能源，产品产量低，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制造具有商业重要性的分子，以便工业可以采用这些方法，从而改善化学过程。酶--自然界的蛋白质催化剂--将被用来代替传统方法，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，并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，这可能会极大地影响它们的行为。 这些知识将用于改进酶，使它们更好地适应化学合成的需要。如果成功的话，这些努力可能会导致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（S4 L）项目，将直接为培养一支高素质的STEM工作队伍做出贡献。该项目的第一部分重点是修饰酶以催化硫代克莱森缩合和β-酮酸脱羧，从而在简单的单反应器系统中产生手性α-氨基-β-羟基酮。第二部分是对映选择性官能团的相互转化。 这集中在一个家庭的酶（老黄酶，OYEs），立体选择性地减少缺电子烯烃。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，但表现出广泛变化的立体选择性，似乎与在催化循环期间改变结构的蛋白质环有关。分子动力学计算表明，环有非常不同的动力学性质，从酶，这可能是孤立的一个氨基酸环。 在该位置的位点饱和诱变将产生突变体，所述突变体将在实验上（关于底物接受性和立体选择性）和计算上（以鉴定动态特性的差异）进行检查。 这个环中的其他位置将通过实验和计算来表征，以更好地定义这个重要的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所起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，有助于可持续的生物制造业。

项目成果

Semi-rational approach to expand the Acyl-CoA Chain length tolerance of Sphingomonas paucimobilis serine palmitoyltransferase

• DOI: 10.1016/j.enzmictec.2020.109515
• 发表时间: 2020-06-01
• 期刊: ENZYME AND MICROBIAL TECHNOLOGY
• 影响因子: 3.4
• 作者: Choe，Hyunjun;Cha，Minsun;Stewart，Jon D.
• 通讯作者: Stewart，Jon D.

Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods

• DOI: 10.1039/c8cy00440d
• 发表时间: 2018-10
• 期刊: Catalysis Science & Technology
• 影响因子: 5
• 作者: Robert W. Powell, III;M. Pilar Buteler;Sunidhi Lenka;M. Crotti;Sara Santangelo;Matthew J. Burg;S. Bruner;E. Brenna;A. Roitberg;Jon D. Stewart

Application of Acetyl-CoA synthetase from Methanothermobacter thermautotrophicus to non-native substrates

• DOI: 10.1016/j.enzmictec.2019.05.005
• 发表时间: 2019-09-01
• 期刊: ENZYME AND MICROBIAL TECHNOLOGY
• 影响因子: 3.4
• 作者: Mouterde, Louis M. M.;Stewart, Jon D.
• 通讯作者: Stewart, Jon D.