LPMO-inspired artificial metalloenzymes for selective oxidation

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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.