Chem.Sci.http://dx.doi.org/10.1039/c4sc01525h(2014)

Artificial metalloenzymes offer the opportunity to marry the versatility of transition metal catalysts with the very high activity and selectivity provided by enzymatic scaffolds. However, designing new metalloenzymes is complicated because transition metals typically require multiple donor atoms to form a stable complex. A single natural amino acid cannot normally provide more than one donor atom and designing new proteins that fold to form a three-dimensional metal binding site is not currently possible. Incorporating unnatural amino acids that contain side-chains capable of binding transition metals into a protein's amino acid sequence offers one solution to this problem.

Credit: © 2014 ROYAL SOCIETY OF CHEMISTRY

To achieve this aim, a team led by Gerard Roelfes at the University of Groningen used the 'amber stop codon suppression' method to site-specifically insert an unnatural amino acid into a protein called 'Lactoccocal multidrug resistance Regulator' (LmrR). In this approach the amber stop codon and an orthogonal tRNA/aminoacyl-tRNA synthase pair are used to incorporate an unnatural amino acid at the position within the protein designated by the amber stop codon. The team used an unnatural amino acid with a 2,2′-bipyridine moiety on its side-chain to create variants of LmrR that were capable of binding Cu2+ ions and were observed to form dimers.

Next, the team investigated whether these artificial metalloproteins were capable of catalysing a Friedel–Crafts alkylation. Variants of LmrR containing bound copper were shown to not only catalyse the reaction but also provide a modest enantiomeric excess. Comparative experiments using a simple copper complex as the catalyst showed no enantiomeric selectivity. To optimize the catalytic properties of the metalloenzyme, the team carried out a mutagenesis study in which amino acid residues surrounding the active site were swapped for alternative amino acids. Successive rounds of mutagenesis produced variants that improved both the yield and the enatioselectivity of the reaction.