Characterizing the link between protein dynamics and catalytic function to improve the design of enzyme biocatalysts

表征蛋白质动力学和催化功能之间的联系，以改进酶生物催化剂的设计

• 批准号: RGPIN-2016-05557
• 负责人: Doucet, Nicolas
• 金额: $ 2.77万
• 依托单位: Institut national de la recherche scientifique
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615767
• 关键词: Characterizing; link; between; protein; dynamics

Enzymes are increasingly being used in industrial settings, primarily as a cost effective, environmentally friendly alternative to harmful chemical compounds. Yet, the engineering of new tailored enzymes dedicated to specific applications remains a very arduous and time consuming endeavor that often yields inefficient biocatalysts. This is mainly attributed to a lack of understanding of how protein engineering affects the 3D structure, catalytic function, and molecular flexibility of enzymes. Recent experimental evidence indicates that several concerted molecular motions occurring on the time scale of the catalytic reaction play an important role in promoting catalysis in numerous enzyme systems. However, we have yet to understand how this atomic flexibility couples to the catalytic event, and whether enzymes with similar structures and/or function also retain comparable molecular motions. Moreover, the effect of the amino acid sequence on the transmission of this dynamic molecular signal remains elusive. To address these critical protein engineering issues, our work will focus on members of the glycoside hydrolase superfamily, which includes an extensive network of distinct biocatalysts involved in the breakdown of biomass for alternative energy sources and for the synthesis of green chemicals. Using an innovative combination of molecular biology techniques and nuclear magnetic resonance (NMR), we will study the effect of single and combinatorial mutations on the molecular flexibility and catalytic function of glycoside hydrolases. By providing clues relating to the modulation of catalytic activity by controlling molecular motions using mutagenesis, our research program has the potential to lead to fundamental breakthroughs in the field of enzyme engineering applied to biocatalysts of significant industrial relevance. Additionally, by providing information on the functional role of conformational exchange in several enzyme systems, the proposed research will offer valuable knowledge on the allosteric control and inhibition of potential protein targets.

酶越来越多地用于工业环境中，主要作为有害化合物的成本效益，环境友好的替代品。然而，专门用于特定应用的新型定制酶的工程设计仍然是一项非常艰巨且耗时的奋进，并且通常会产生低效的生物催化剂。这主要是由于缺乏对蛋白质工程如何影响酶的3D结构，催化功能和分子灵活性的理解。最近的实验证据表明，在催化反应的时间尺度上发生的几个协同分子运动在促进许多酶系统的催化中起着重要的作用。然而，我们还没有了解这种原子的灵活性如何耦合到催化事件，以及具有相似结构和/或功能的酶是否也保留了可比的分子运动。此外，氨基酸序列对这种动态分子信号传递的影响仍然难以捉摸。为了解决这些关键的蛋白质工程问题，我们的工作将集中在糖苷水解酶超家族的成员，其中包括一个广泛的网络，涉及生物质的替代能源和绿色化学品的合成分解不同的生物催化剂。使用分子生物学技术和核磁共振（NMR）的创新组合，我们将研究单个和组合突变对糖苷水解酶的分子灵活性和催化功能的影响。通过使用诱变控制分子运动提供有关催化活性调节的线索，我们的研究计划有可能导致酶工程领域的根本性突破，应用于具有重要工业意义的生物催化剂。此外，通过提供关于构象交换在几种酶系统中的功能作用的信息，拟议的研究将提供关于潜在蛋白质靶点的变构控制和抑制的有价值的知识。