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

酶越来越多地用于工业和制药环境，主要是作为一种成本效益高、对环境友好的有害有机溶剂的替代品。然而，专门用于特定用途的新酶的工程设计仍然是一项非常艰巨和耗时的努力，通常会产生低效的生物催化剂。这主要是由于缺乏对酶工程如何影响酶的三维结构、催化功能和分子柔韧性的理解。最近的实验证据表明，在催化反应的时间尺度上发生的几种协调一致的分子运动在许多酶系统中起着促进催化作用的重要作用。然而，我们尚未了解这种原子柔韧性如何与催化事件耦合，以及具有相似结构和/或功能的酶是否也保持类似的分子运动。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，其中包括广泛的不同生物催化剂网络，这些生物催化剂涉及杀菌、血管生成、细胞毒性和抗肿瘤活性。利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核糖核酸酶分子柔韧性和催化功能的影响。通过利用诱变控制分子运动来调节催化活性，我们的研究项目有可能在酶工程应用于工业和制药相关的生物催化剂领域取得根本性突破。此外，通过表征具有关键细胞功能的几种核糖核酸酶的催化分子运动，该研究将为这些临床相关靶点的潜在变构调节提供有价值的信息。

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.