Artificial metalloenzymes : modified proteins as tuneable transition metal catalysts and their application in oxidative lignin degradation

Abstract

The selective oxidation of organic molecules is fundamentally important to life and immensely useful in industry.

Metalloenzyme catalysed oxidations often display exquisite substrate specificity as well as regio and/or stereoselectivity. Huge strides have occurred in the field of biocatalysis in recent years. Work has developed by taking inspiration from nature’s enzymes, to use directed evolution and engineering methods to create tailor made catalysts. Artificial Metalloenzymes (ArMs) provide the possibility to expand this repertoire further by combining the advantageous features of enzymes with the versatile reaction scope of transition metals.

The initial chapter in this thesis takes a look into recent literature about artificial metalloenzymes and their application in oxidation catalysis.

Chapter two describes the design rationale and synthesis of protein templates and synthetic cofactors for the development of artificial metalloenzymes. Successful modification was achieved for a wide library of nitrogen donor ligands, creating an array of artificial metalloenzymes that can be tested in catalytic reactions. In the absence of a crystal structure of the modified protein, UV and CD analysis were carried out to gather characteristic information about the artificial metalloenzymes and their metal binding properties. An investigation was also carried out to determine the most accurate method to calculate protein concentration once it has been modified with a cofactor.

The third chapter describes the application of protein engineering to increase the thermostability of the target protein. Variants of an artificial metalloenzyme were created by rational design using structural and bioinformatic information. The variants were tested to identify mutations that enhanced the stability of the protein scaffold. Significant increases in melting temperature were observed in a number of the modified metalloenzymes. Their ability to withstand higher reaction temperatures resulted in increased activity in the hydroformylation of 1-octene, with >5-fold improvements in turnover numbers (TON).

The fourth chapter reports the use of artificial metalloenzymes in oxidation catalysis. In particular their application to the degradation of lignin is investigated. Using a model compound that mimics the most abundant linkage within lignin as a substrate, a wide array of artificial metalloenzymes were tested to study if any oxidation or cleavage occurs. Investigations were carried out to find the optimum conditions varying catalyst loading and buffer/solvent composition. Complete selective conversion to ketone product is observed using SCP-2L A100C modified with a tris(2-pyridylmethyl) amine based cofactor, coordinated to Fe(OTf)₂.2MeCN. Engineering the protein scaffold to incorporate glutamic acid was found to improve the ArM activity, showing that rational design of the protein environment using metal binding amino acids can be a method to improve the overall activity of an artificial metalloenzyme.