The use of enzymes for the synthesis of organic chemicals depends on their ability to perform reactions outside the scope they evolved for.
We are interested in the design and engineering of enzymes, either to be integrated in metabolic pathways or to be used as isolated entities outside the cells.
We use computational and modelling techniques to study substrate binding, catalytic mechanisms and protein stability, and to predict beneficial mutations.
We apply genetic and chemical modification techniques, in order to modify enzyme functionality for a range of applications in synthetic biology, biocatalysis and healthcare technologies.
Our goal is to integrate the transformed enzymes within industrial biotechnological processes, to deliver a sustainable alternative for the production of high value chemicals.
Design of artificial metalloenzymes for non-natural transformations
Artificial metalloenzymes are built by chemical modification of proteins with catalytic functionalities that are not available in nature, thus expanding the catalytic scope of enzymes towards non-natural activities. Their active site consists of a hydrophobic pocket, which is large enough to accommodate a chemical catalyst and a non-natural substrate.
We develop computational and experimental strategies in order to improve the design of artificial metalloenzymes. We use alcohol dehydrogenases (ADHs) as protein scaffolds, due to their naturally evolved capacity to stabilise hydride transfer transition states. Synthetic transition metal catalysts can be positioned in the proximity of the active site, by covalent modification of ADH mutants possessing single cysteines. Both chemical and genetic optimisation of the artificial metalloenzymes can be performed.
Engineering of carbonic anhydrase
The aim of this project is to design an ultra-thermostable carbonic anhydrase with promiscuous reactivity towards the reduction of carbon dioxide. We use modelling techniques to understand the thermostability of carbonic anhydrase, and to design and engineer stabilising mutations. The catalytic zinc ion from the active site of the thermostable enzyme can be replaced with other transition metals, which have been reported to reduce carbon dioxide into formate. The natural ability of TaCA to bind CO2 can thus be combined with the non-natural activity of the transition metal, to provide an artificial metalloenzyme for carbon dioxide utilisation.
Radical SAM enzymes for biotechnology
Radical SAM enzymes are a family of enzymes that harness free radical chemistry in their catalysis for the synthesis of a very broad spectrum of valuable chemicals. Biotechnological processes using these enzymes offer opportunities to combine sustainable chemistry approaches with the high potential of free radicals – chemical intermediates which are difficult to control in industrial processes, but which nature has been able to harness to produce valuable materials, including antibiotics, anticancer, and antiviral drugs.
We aim to better understand key catalytic features of these enzymes, in order to harness this information to re-engineer the enzymes to increase catalytic turnover and change their substrate scope. Current projects deal with the rSAM enzyme QueE involved in biosynthesis of nucleotide analogue antibiotics, the biosynthesis of Biotin (Vitamin B7), lysine amino mutase (LAM), and the pyruvate formate lyase activating enzyme (PFL-AE). Along with engineering their catalysis we are particularly interested in understanding oxygen sensitivity of these enzymes and factors influencing the redox chemistry of the central inorganic iron sulphur clusters.