Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.

Artificial metalloenzymes for asymmetric catalysis were created by in vivo incorporation of unnatural metal binding amino acids into LmrR.

The emergence of robust methods to expand the genetic code allows incorporation of noncanonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chemical functionalities in enzymes. In this perspective, we show how this powerful methodology is used to create enzymes with improved and novel, even new-tonature, catalytic activities. We provide an overview of the current state of the start and discuss the potential benefits of developing and using enzymes with genetically encoded noncanonical amino acids compared to enzymes containing canonical amino acids only.

Starting from biochemical knowledge followed by computational design, an artificial metallo-hydratase comprising an unnatural metal binding amino acid was created.