Dr Michèle Salmain from Université Pierre et Marie Curie, Paris gives an overview of the process involved in creating artificial enzymes
Artificial enzymes or ‘artzymes’ are man-made constructs in which an active site is implanted within a protein host to endow it with a (new) catalytic activity. In this field, we are specifically looking to create artificial metalloenzymes by incorporating metallic species into protein hosts to drive certain reactions to high enantioselectivity and chemoselectivity under environmentally friendly conditions.
The reaction that we are focusing on is the reduction of ketones to produce secondary alcohols using a reaction coined ‘asymmetric transfer hydrogenation’ that is catalysed by ruthenium and rhodium complexes. This reaction makes use of formate as a hydrogen source and can be carried out under smooth conditions in the water.
During the design of these novel biocatalysts, great attention was paid to 3 parameters. The first was the protein scaffold, which was selected based on stability, availability and the presence of a binding pocket in which the reaction could proceed. Second, the method of incorporating the metallic entity was considered, and finally, we examined the catalytic activity that the transition metal centre would likely confer to the hybrid. These parameters are highly interdependent, as the surrounding protein environment affects catalysis and how well the metal complex is incorporated.
Initially, the protein papain was selected as the scaffold. Owing to its native activity and 3D structure, this protein is amenable to the covalent anchoring approach. We successfully produced several metallopapains with the targeted catalytic activity, but the resulting enantiomeric excesses were only modest.
Latterly, we selected beta-lactoglobulin, a protein present in the whey fraction of cows’ milk. Beta-lactoglobin has a peculiar 3D structure (known as beta-barrel) since it folds like a calyx. Interestingly, it can bind fatty acids with high affinity and once bound the carboxylate group of the fatty acids is positioned at the entrance of the calyx while the alkyl chain occupies the hydrophobic tunnel. It was anticipated that appending the metal centre to the carboxylate group of fatty acids might further allow tight control of its positioning with respect to the protein environment.
This kind of process is called supramolecular anchoring. Luckily enough circular dichroism studies showed that some of the metal cofactors synthesised from saturated fatty acids did associate with beta-lactoglobulin. This finding was substantiated by X-ray crystallographic studies that confirmed the metal complex was indeed located at the entrance of the calyx.
After these steps, we tested the catalytic activity of the hybrids on a standard hydrogenation reaction by measuring the conversion rate of the substrate and the enantiomeric excess with high-pressure liquid chromatography. Some hybrids gave quantitative conversion rates and other hybrids gave enantiomeric excess up to 32%. For comparison, the popular, ruthenium-based Noyori’s catalyst afforded an enantiomeric excess of only 27%.
It is now planned to expand the approach to the design of artificial copper enzymes. Further to this, we are looking to understand the reasons why some of these artificial enzymes have such selectivity and reactivity while others do not, which should, in turn, improve the artzyme design process.
Dr Michèle Salmain
Université Pierre et Marie Curie, Paris
Tel: +33 (0) 1 44 27 67 32
michele.salmain@upmc.fr
