Exploiting Non‐Canonical Amino Acids to Alter Enzyme Substrate Specificity

Enzymes are constructed from the 20 proteogenic amino acids, but Nature has expanded the chemistries available in proteins by recruiting cofactors or inserting non‐canonical amino acids (ncAAs). Until recently, protein engineering was restricted to using the 20 proteogenic amino acids. Using a chemical method, we are able to produce proteins containing ncAAs quickly and in high yields. This method works by first converting a cysteine into a dehydroalanine intermediate, before reacting with a thiol to produce the non‐canonical side chain. Initial studies focussed on replacing the catalytic lysine in the aldolase N ‐acetyl‐neuraminic acid lyase (NAL) with a γ‐thialysine, this enzyme has been characterised both kinetically and crystallographically. Using this methodology, we have explored the effect of an increased amino acid repertoire on enzyme substrate specificity by inserting multiple ncAAs (~16) at various positions (~11) throughout the active site of NAL, and screening for altered activity. This has produced an ncAA‐containing aldolase with a 10 fold increased k cat , with a shortened substrate, over that of the wild‐type. Saturation mutagenesis has also revealed that this increase in activity is not achievable with any of the proteogenic amino acids. Crystallographic and computational studies have been used to elucidate the mechanism by which this ncAA increases activity. This work highlights that activities previously thought inaccessible by use of the 20 proteogenic amino acids may now be achievable by use of ncAAs.