21BBSRC-NSF/BIO - Evolving quantum mechanical tunnelling in enzymes

21BBSRC-NSF/BIO - 酶中不断发展的量子力学隧道

• 批准号: BB/X000974/1
• 负责人: Sam Hay
• 金额: $ 62.75万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX000974%2F1
• 关键词: 21BBSRC; NSF; BIO; Evolving; quantum

H-transfer reactions involving hydride, hydrogen atom or proton transfers are ubiquitous in enzyme-catalysed reactions. It is now well established that these reactions can occur via a mechanism involving some degree of quantum mechanical tunnelling (QMT), and thus are a feature of quantum biology. The QMT contribution is highly variable and an open question remains whether there has been evolutionary pressure to select for QMT during enzyme catalysis. Theoretically, it is possible to increase the rate of an enzyme-catalysed H-transfer reaction by increasing the tunnelling contribution, so this could offer a fitness advantage. However, typical strategies to increase the tunnelling contribution are also likely to increase the rate of classical (over-the-barrier) H-transfer. In this proposal we aim to combine experimental studies of H-tunnelling in enzyme catalysed reactions with directed (laboratory) evolution (DE) and computational chemistry. We will study two unrelated enzymes in parallel: (i), alcohol dehydrogenase (ADH) is a well-studied model hydride-tunnelling and industrial biocatalysis enzyme.(ii), MBHase is a de novo Morita-Baylis-Hillman enzyme, that we have recently developed through DE.DE will be performed using high- and medium-throughput screening methods that select for activity with both protiated and deuterated substrates (so either H- or D- transfer occurs) in order to allow parallel evolution of enzymes evolved specifically for H- and D- transfer. Selected variants will be experimentally characterised using additional kinetics methods (stopped-flow pre-steady state rate constants and kinetics isotope effects, and temperature dependencies) and by X-ray crystallography to solve structures where possible. These selected variants will also be investigated using computational chemistry, which will allow the QMT contribution to be determined. This approach will allow a new and more comprehensive approach to the exploration of the relationships between H- and D- transfer kinetics and QMT during enzyme evolution, and will establish whether selecting for improved D-transfer kinetics provides new avenues for improving enzyme performance.We will take advantage of our recent breakthroughs in correlating computed QMT contributions with experimental kinetics isotope effects (ratio of H- and D-transfer kinetics), in evolving MBHase and in the development of the free energy QM/MM computational methods of characterising enzyme-catalysed reaction chemistry. To the best of our knowledge, this is the first attempt at using DE to probe QMT in enzymes, or to use deuterium kinetics as selection during DE experiments. A recent paper showed improvement in a laboratory evolved enzyme for deuteration of short chain acids, but selection was performed with protium, not deuterium. The project is thus both timely and novel and will provide new insight into the role of QMT during evolution of H-transfer enzymes. Further, as efficient routes to selective deuteration of pharmaceuticals is currently a hot topic, and many industrially-important enzymes catalyse H-transfers, the methodology also promises to lead to a new approach to enzyme optimisation for applications in synthesis and industrial biocatalysis.

涉及氢化物、氢原子或质子转移的氢转移反应在酶催化反应中普遍存在。现在已经确定，这些反应可以通过涉及一定程度的量子力学隧道效应（QMT）的机制发生，因此是量子生物学的一个特征。QMT的贡献是高度可变的，一个悬而未决的问题仍然是是否有进化的压力，选择QMT在酶催化。从理论上讲，通过增加隧道效应的贡献可以提高酶催化的氢转移反应的速率，因此这可以提供适合度优势。然而，增加隧穿贡献的典型策略也可能增加经典（过势垒）H-转移的速率。在这个建议中，我们的目标是结合联合收割机实验研究的H-隧道在酶催化反应与定向（实验室）进化（DE）和计算化学。我们将平行研究两种不相关的酶：（i）乙醇脱氢酶（ADH）是一种研究得很好的模型生物隧道和工业生物催化酶。(ii)MBHase是我们最近通过DE开发的从头Morita-Baylis-Hillman酶。DE将使用高通量和中等通量筛选方法进行，该方法选择质子化和氘代底物的活性（因此发生H-或D-转移），以允许特异性进化用于H-和D-转移的酶的平行进化。选择的变体将使用其他动力学方法（停流前稳态速率常数和动力学同位素效应以及温度依赖性）进行实验表征，并通过X射线晶体学在可能的情况下解析结构。还将使用计算化学研究这些选定的变体，这将允许确定QMT贡献。这种方法将允许一种新的和更全面的方法来探索酶进化过程中H-和D-转移动力学与QMT之间的关系，我们将利用我们最近在将计算的QMT贡献与实验动力学同位素效应相关联方面的突破，（H-和D-转移动力学的比率），在进化MBHase和在表征酶催化反应化学的自由能QM/MM计算方法的发展中。据我们所知，这是第一次尝试使用DE探测酶中的QMT，或使用氘动力学作为DE实验期间的选择。最近的一篇论文表明，在实验室进化的短链酸的氘化酶的改进，但选择进行了与protium，而不是氘。因此，该项目既及时又新颖，将为QMT在H-转移酶进化过程中的作用提供新的见解。此外，由于药物选择性氘化的有效途径是目前的热门话题，并且许多工业上重要的酶催化H-转移，该方法还有望导致用于合成和工业生物催化的酶优化的新方法。

项目成果

Strategies for designing biocatalysts with new functions

设计具有新功能的生物催化剂的策略

• DOI: 10.1039/d3cs00972f
• 发表时间: 2024
• 期刊: Chemical Society Reviews
• 影响因子: 46.2
• 作者: Bell E