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

酶正越来越多地被用于工业环境中，主要是作为有害化合物的一种成本效益和环境友好的替代品。然而，设计专门用于特定应用的新的量身定做的酶仍然是一项非常艰巨和耗时的努力，往往会产生效率低下的生物催化剂。这主要归因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了解。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的分子运动对许多酶系统中的催化起着重要的促进作用。然而，我们还不知道这种原子的灵活性是如何耦合到催化事件上的，以及具有相似结构和/或功能的酶是否也保持着类似的分子运动。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上，其中包括参与分解生物质以替代能源和合成绿色化学品的广泛的不同生物催化剂网络。利用分子生物学技术和核磁共振技术的创新组合，我们将研究单一突变和组合突变对糖苷水解酶分子柔性和催化功能的影响。通过提供有关利用突变控制分子运动来调节催化活性的线索，我们的研究计划有可能在应用于具有重大工业意义的生物催化剂的酶工程领域取得根本性突破。此外，通过提供有关构象交换在几个酶系统中的功能作用的信息，拟议的研究将为潜在的蛋白质靶标的变构控制和抑制提供有价值的知识。

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.