In silico enzymology: A mechanistic study of prenylated flavin-dependent enzymes

计算机酶学：异戊二烯化黄素依赖性酶的机理研究

• 批准号: 2449591
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2449591
• 关键词: silico; enzymology; mechanistic; study; prenylated

Many enzymes make use of cofactors to aid in catalysis, as they offer alternative reactivities to the canonical amino acids found in the enzyme active site. One such cofactor is the recently discovered prenylated flavin (prFMN), which is used in the UbiD family of enzymes to catalyse reversible decarboxylation of a range of substrates; many of which are of interest to the bioenergy and biotechnology industries. This project will use a range of computational chemistry approaches, including density functional theory (DFT) modelling and molecular dynamics (MD) simulations, to study the mechanism of prFMN-dependent enzymes. Of particular interest is the reversible 1,3-dipolar cycloaddition thought to be crucial to catalysis, and the reversible (de)carboxylation that occurs readily under atmospheric CO2. In order to benchmark these calculations, the project will also include an experimental component, comprising enzyme kinetic measurements made using steady state and rapid-mixing methods and kinetic isotope effect (KIE) measurements made using NMR. An element of both computational and experimental method development will be required and it is envisaged an iterative compute-test cycle will be used to validate proposed mechanism. Once the mechanism of wild-type enzyme(s) is established, the approach will be expanded to identify related enzymes with new substrate profiles and/or to improve the catalytic performance of established enzymes and to expand their substrate scope. The synthesis of prFMN by UbiX enzymes will also be investigated to explore the potential of the biosynthesis of new prFMN-like cofactors, which may expand the reactivity of prFMN-dependent enzymes.The project is a computationally-driven study of a family of biotechnologically-important enzymes. It falls within the remit of both 'technologies and methodological development' and 'molecules, cells and industrial biotechnology' and is firmly embedded at the interface of chemistry, biology and physics, a key driver for BBSRC in the 'Exploiting new ways of working' agenda. It draws on the core bioscience skills of bioinformatics and mathematics, and provides advanced research training in (biologically-relevant) computational chemistry, recombinant protein production and biophysical characterisation and NMR spectroscopy. Overall, the work will provide a highly interdisciplinary approach to (bio)chemistry/ biophysics-based research, offering highly diverse training opportunities to a PhD student, who will have the additional benefit of being able to access the supervisors' laboratories on a daily basis, as they are all co-located within the same building, the Manchester Institute of Biotechnology (MIB).

许多酶利用辅因子来帮助催化，因为它们提供与酶活性部位中的典型氨基酸相反的反应活性。一种这样的辅因子是最近发现的戊烯基黄素(PrFMN)，它在UbiD酶家族中用于催化一系列底物的可逆脱羧化；其中许多对生物能源和生物技术行业感兴趣。本项目将使用一系列计算化学方法，包括密度泛函理论(DFT)建模和分子动力学(MD)模拟，来研究prFMN依赖的酶的机制。特别令人感兴趣的是被认为对催化至关重要的可逆1，3-偶极环加成反应，以及在大气二氧化碳下很容易发生的可逆(去)羧化反应。为了作为这些计算的基准，该项目还将包括一个实验部分，包括使用稳态和快速混合方法进行的酶动力学测量以及使用核磁共振进行的动力学同位素效应测量。将需要计算和实验方法开发的一个要素，预计将使用迭代计算-测试周期来验证所提议的机制。一旦建立了野生型酶(S)的机制，该方法将得到扩展，以识别具有新底物图谱的相关酶和/或改进已建立的酶的催化性能并扩大其底物范围。UbiX酶合成prFMN也将被研究，以探索新的prFMN样辅因子的生物合成潜力，这可能会扩大prFMN依赖的酶的反应活性。该项目是对一系列具有重要生物技术意义的酶的计算驱动的研究。它属于“技术和方法开发”和“分子、细胞和工业生物技术”的范畴，并牢固地植根于化学、生物和物理的交界处，这是BBSRC在“探索新的工作方式”议程中的关键驱动力。它利用生物信息学和数学的核心生物科学技能，并提供(与生物相关的)计算化学、重组蛋白质生产、生物物理表征和核磁共振光谱学方面的高级研究培训。总体而言，这项工作将为(生物)化学/生物物理学基础研究提供高度跨学科的方法，为博士生提供高度多样化的培训机会，他们将获得额外的好处，能够每天访问主管的实验室，因为他们都位于同一栋建筑内，即曼彻斯特生物技术研究所(MIB)。