SusChem: New Methodologies in Biocatalysis

SusChem：生物催化新方法

基本信息

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

项目摘要

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.

使用传统化学方法，生产和纯化高价值分子（香料、香料、染料、抗生素）通常会消耗大量能源，产品产量低，并产生大量危险废物。该项目的目标是使用最大限度减少浪费和最大限度提高产量的策略来制造商业上重要的分子，以便工业界可以采用这些方法，从而改进化学工艺。酶——大自然的蛋白质催化剂——将取代传统方法，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起作用，并且它们是完全可生物降解的。除了开发新的反应策略外，酶的三维结构也将被确定。计算机建模将表明它们的形状如何变化，这会极大地影响它们的行为。这些知识将用于改进酶，使它们更适合化学合成的需要。如果成功，这些努力可能会带来更加可持续和环境友好的化学工业。 PI 积极参与佛罗里达大学的生命科学 (S4L) 项目，将直接有助于培养高素质的 STEM 劳动力队伍。该项目的第一部分重点是修改酶以催化硫代克莱森缩合和 β-酮酸脱羧，以便在简单的单反应器系统中产生手性α-氨基-β-羟基酮。对映选择性官能团互变是第二部分的主题。该研究重点关注立体选择性还原缺电子烯烃的酶家族（老黄酶，OYE）。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，这似乎与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，不同酶的环具有非常不同的动态特性，这可能被隔离为环中的单个氨基酸。该位置的位点饱和诱变将产生突变体，突变体将通过实验（关于底物接受性和立体选择性）和计算（以识别动态特性的差异）进行检查。该环中的其他位置将通过实验和计算来表征，以更好地定义这一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的作用的基本了解将极大地扩展设计具有所需功能的蛋白质的能力，从而为可持续的生物制造行业做出贡献。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

嗜热甲烷杆菌乙酰辅酶A合成酶在非天然底物中的应用

DOI：
10.1016/j.enzmictec.2019.05.005
发表时间：
2019-09
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.

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

DOI：
10.1039/c8cy00440d
发表时间：
2018-10-01
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
通讯作者：
Jon D. Stewart

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

扩大稀动鞘氨醇单胞菌丝氨酸棕榈酰转移酶酰基辅酶A链长度耐受性的半理性方法