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

许多酶利用辅助因子来辅助催化，因为它们提供了在酶活性位点发现的规范氨基酸的替代反应性。一个这样的辅助因子是最近发现的preylated flavin (prFMN)，它在UbiD酶家族中用于催化一系列底物的可逆脱羧；其中许多是生物能源和生物技术行业感兴趣的。该项目将使用一系列计算化学方法，包括密度泛函数理论（DFT）建模和分子动力学（MD）模拟，来研究依赖prfmn的酶的机制。特别令人感兴趣的是对催化作用至关重要的可逆1,3-偶极环加成反应，以及在大气CO2下容易发生的可逆（脱）羧化反应。为了对这些计算进行基准测试，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（KIE）测量。将需要计算方法和实验方法两方面的发展，预计将采用迭代计算测试周期来验证所提议的机制。一旦确定了野生型酶的机制，该方法将扩展到鉴定具有新底物特征的相关酶和/或提高已建立酶的催化性能并扩大其底物范围。我们还将研究UbiX酶对prFMN的合成，以探索生物合成新的prFMN样辅助因子的潜力，这可能会扩大prFMN依赖性酶的反应性。该项目是一项计算驱动的对生物技术重要酶家族的研究。它属于“技术和方法发展”和“分子，细胞和工业生物技术”的范围，并牢固地嵌入化学，生物学和物理学的界面，这是BBSRC在“开发新的工作方式”议程中的关键驱动因素。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，重组蛋白质生产和生物物理表征和核磁共振光谱的高级研究培训。总的来说，这项工作将为基于（生物）化学/生物物理学的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，他们将有额外的好处，可以每天访问导师的实验室，因为他们都位于同一栋建筑内，曼彻斯特生物技术研究所（MIB）。

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).