Enzyme cooperativity, biocatalysis and bioconjugation

酶协同作用、生物催化和生物结合

• 批准号: RGPIN-2017-04107
• 负责人: Auclair, Karine
• 金额: $ 3.28万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710456
• 关键词: Enzyme; cooperativity; biocatalysis; bioconjugation

Enzymes are not only fascinating catalysts, but they are also the target of most drugs. Thanks to their complex 3D structure, enzymes show many interesting characteristics rarely found in chemical catalysts. Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous enzymes found across all kingdoms and involved in processes as varied as drug metabolism, biosynthesis of natural products, steroids, lipids, and more. Some P450s (e.g. CYP3A4) are known to show allosteric effects and/or use a unique cooperativity mechanism involving direct interactions between multiple copies of the same or different ligands bound to the enzyme at the same time. Such a behavior is quite challenging to delineate using existing methods. In order to study the allostery and cooperativity of CYP3A4, we have developed a new bioconjugation approach and a number of biophysical methods which will be applied. We are also currently developing a kinetic method using isothermal calorimetry. The use of enzymes in synthetic chemistry is advantageous not only because it is compatible with environmentally benign conditions but also because many enzymes catalyze reactions that are inaccessible to chemical catalysts. For example, P450s are of interest to us because of their ability to catalyze selective hydroxylations at unactivated C-H bonds. Chitinases and lignolytic peroxidases are also of interest to us because they can degrade some of the toughest and most abundant substrates, chitin and lignin respectively. We propose to develop new methods to facilitate biocatalytic applications. For example, we have pioneered the use of chemical auxiliaries to control and predict the regio- and stereo-selectivity of P450-catalyzed reactions. Using an enzyme with a large promiscuous substrate binding pocket, we have recently demonstrated that constraining the active site via rational mutagenesis can dramatically shift the selectivity of the enzyme. Finally, we have gathered preliminary data demonstrating that mechanochemistry can positively affect enzymatic processes and even surpass current chemical industrial processes. We propose to apply and optimize these methods for specific processes of interest. The overall goals of this research program are to develop new tools to study enzymes and apply these in enzymology and to facilitate biocatalysis. Representative specific aims include: - Apply our biophysical methods to study aminoglycoside 3'-phosphoryltransferase - Study P450 using isothemal calorimetry in kinetics mode - Use our new CYP3A4 bioconjugates and generate new ones to study CYP3A4 allostery and cooperativity - Identify new chemical auxiliaries to control the regio- and stereo-selectivities of P450s - Apply, optimize and investigate our mechanochemical method with new enzymatic reactions

酶不仅是迷人的催化剂，而且也是大多数药物的靶点。由于其复杂的3D结构，酶显示出许多在化学催化剂中很少发现的有趣特性。 细胞色素P450酶（CYP或P450）是在所有王国中发现的普遍存在的酶，并参与药物代谢，天然产物，类固醇，脂质等生物合成的过程。已知一些P450（例如CYP 3A 4）显示变构效应和/或使用独特的协同机制，涉及同时与酶结合的相同或不同配体的多个拷贝之间的直接相互作用。使用现有方法描绘这样的行为是相当具有挑战性的。为了研究CYP 3A 4的变构性和协同性，我们开发了一种新的生物偶联方法和一些生物物理学方法。我们目前还在开发一种使用等温量热法的动力学方法。 在合成化学中使用酶是有利的，不仅因为它与环境友好的条件相容，而且因为许多酶催化化学催化剂无法达到的反应。例如，我们对P450感兴趣，因为它们能够催化未活化的C-H键的选择性羟基化。几丁质酶和木质素分解过氧化物酶也是我们感兴趣的，因为它们可以分别降解一些最坚韧和最丰富的底物，几丁质和木质素。我们建议开发新的方法，以促进生物催化应用。例如，我们率先使用化学助剂来控制和预测P450催化反应的区域和立体选择性。使用一个大的混杂底物结合口袋的酶，我们最近已经证明，通过合理的诱变限制活性位点可以显着改变酶的选择性。最后，我们已经收集了初步数据，证明机械化学可以积极影响酶促过程，甚至超过目前的化学工业过程。我们建议应用和优化这些方法的特定过程的利益。 该研究计划的总体目标是开发新的工具来研究酶，并将其应用于酶学，促进生物催化。代表性的具体目标包括： - 应用我们的生物物理方法研究氨基糖苷类3 '-磷酸转移酶 - 在动力学模式下使用等温量热法的研究P450 - 使用我们新的CYP 3A 4生物缀合物并产生新的缀合物来研究CYP 3A 4变构和协同性 - 确定新的化学助剂以控制P450的区域和立体选择性 - 应用，优化和研究我们的机械化学方法与新的酶促反应