LPMO-inspired artificial metalloenzymes for selective oxidation

生物催化正在迅速崛起，成为一系列化学工业的一种强大而广泛的工具--从合成复杂和高度功能化的药物到大量生产生物燃料。生物催化的流行源于设计和制造定制催化剂的能力不断增强，这些催化剂能够进行一系列理想的转化，具有现有化学方法无法比拟的反应性和选择性。其中一种转化是有机分子的选择性氧化-这一反应每年用于生产数百万吨的醇、羰基和环氧化物前体，但仍远未得到优化。由于对环境的日益关注，对催化方法学的需求从未像现在这样强烈，生物催化可能会提供一个最佳的解决方案。化学方法正在发展，但仍然需要苛刻的条件、高温和经常出现被过度氧化的杂质的问题，而酶可以在水中、在环境条件下以及对所需化合物的完全选择性地执行相同的反应。溶解多糖单加氧酶(LPMOS)是一类铜基金属酶，于2010-2011年被发现，它能够执行多糖中发现的C-H键的选择性氧化。它们独特的活性部位是由两个组氨酸残基配位的铜离子形成的，其中一个残基是蛋白质的氨基末端，这种排列通常被称为“组氨酸支架”。这些酶不仅彻底改变了生物燃料生产领域，而且还改变了铜催化氧化在自然界中发生的概念。铜的活性部位被发现在蛋白质的一个非常暴露的溶剂表面，这是多糖活性的完美选择，但阻止了这些酶在小分子的选择性氧化中有效。本项目的目标是生产和表征LPMO启发的人造金属酶，并评估它们对小分子底物的氧化能力。在加入铜离子之前或之后，能够模仿希氏支架排列的配体将被合成、表征并偶联到工程球状蛋白上。这样形成的人造金属蛋白将通过各种技术进行表征，如X射线结晶学、EPR光谱和质谱学。酶活性和氧化能力将通过在一系列可能的底物上进行分析来评估。随后将进行进一步的工程和表征循环，以调整金属酶的性质和底物范围。成功的候选人将获得蛋白质生产、纯化和工程、光谱学(例如EPR、核磁共振)、结构生物学和生化分析方面的技能。该项目的应聘者将拥有化学、生物化学或自然科学学位，对生物无机/生物化学有浓厚兴趣，并将致力于一个多学科项目，获得各种化学和生物化学技术方面的经验。

Biocatalysis is rapidly emerging as a powerful and widespread tool for a range of chemical industries - from the synthesis of complex and highly functionalised pharmaceuticals to the bulk production of biofuels. The popularity of biocatalysis stems from an ever-increasing ability to design and manufacture bespoke catalysts which are able to carry out a range of desirable transformations with reactivity and selectivity which are unrivalled by existing chemical methods.One such transformation is the selective oxidation of organic molecules - a reaction which is used to produce millions of tons of alcohols, carbonyl- and epoxide precursors each year in all areas of chemical industry, but is still far from optimised. As a consequence of growing environmental concerns, the need for a catalytic methodology has never been greater and biocatalysis may present an optimal solution. Chemical methods are evolving but still require harsh conditions, high temperatures and often present the problem of over-oxidised impurities, whereas enzymes can perform the same reactions in water, under ambient conditions and with complete selectivity to the desired compound.Lytic polysaccharide monooxygenases (LPMOs) are a class of Cu-based metalloenzymes, discovered in 2010-2011, which are able to perform the selective oxidation of C-H bond found in polysaccharides. Their unique active site is formed by a copper ion coordinated by two histidine residues, one of which being the amino-terminus of the protein, in an arrangement commonly referred to as the "Histidine brace". These enzymes have not only revolutionised the field of biofuel production, but also transformed the notion of how Cu-catalysed oxidations occur in Nature. The copper active site is found on a very solvent exposed surface of the protein, which is perfect for polysaccharide activity, but prevents these enzymes to be effective in the selective oxidation of small molecules.The objectives of this project will be to produce and characterise LPMO-inspired artificial metalloenzymes, and to evaluate their oxidative abilities towards small molecule substrates. Ligands able to mimic the His brace arrangement will be synthesised, characterised and coupled to an engineered globular protein, before or after addition of Cu ions. The so-formed artificial metalloprotein will be characterised by a variety of techniques, such as X-ray crystallography, EPR spectroscopy and mass spectrometry. The enzymatic activity and oxidative abilities will be evaluated by assays on an array of possible substrates. Further cycles of engineering and characterisation will then be performed to tune the properties and the substrate scope of the metalloenzyme.The successful candidate will gain skills in protein production, purification and engineering, spectroscopy (e.g. EPR, NMR), structural biology and biochemical assays.Candidates for this project will have a Chemistry, Biochemistry or Natural Sciences degree with a strong interest in bioinorganic/biological chemistry and will be working on a multidisciplinary project, gaining experience in a wide variety of Chemistry and Biochemistry techniques.