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和制药环境，主要是作为有害有机溶剂的一种成本效益和环境友好的替代品。然而，专门用于特定应用的新酶的工程仍然是一项非常艰巨和耗时的努力，往往产生效率低下的生物催化剂。这主要归因于缺乏对酶工程如何影响酶的三维结构、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尚不清楚。为了解决这些关键的酶工程问题，我们的工作将集中在核糖核酸酶超家族的成员上，其中包括参与杀菌、血管生成、细胞毒和抗肿瘤活动的不同生物催化剂的广泛网络。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核糖核酸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工业和制药相关生物催化剂的酶工程领域取得根本性突破。此外，通过表征在几种具有关键细胞功能的核糖核酸酶中催化作用的分子运动，拟议的研究将提供关于这些临床相关靶标潜在变构调节的有价值的信息。

Enzymes are increasingly being used in industrial and pharmaceutical settings, primarily as a cost effective, environmentally friendly alternative to harmful organic solvents. Yet, the engineering of new enzymes dedicated to specific applications remains a very arduous and time consuming endeavour that often yields inefficient biocatalysts. This is mainly attributed to a lack of understanding of how enzyme engineering affects the 3D structure, catalytic function, and molecular flexibility of enzymes. Recent experimental evidence indicates that several concerted molecular motions occurring on the timescale 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 unknown. To address these critical enzyme engineering issues, our work will focus on members of the ribonuclease superfamily, which includes an extensive network of distinct biocatalysts involved in bactericidal, angiogenic, cytotoxic and anti-tumoural activities. Using an innovative combination of molecular biological techniques and nuclear magnetic resonance (NMR), we will study the effect of single and combinatorial mutations on the molecular flexibility and catalytic function of ribonucleases. 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 industrial and pharmaceutical relevance. Additionally, by characterizing molecular motions that contribute to catalysis in several ribonucleases with critical cellular function, the proposed research will provide valuable information on the potential allosteric modulation of these clinically relevant targets.