An evolutionary path: from hydrolase to ligase by substitution

The understanding of how proteins evolve to perform novel function has long been sought by biologists. Two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze distinct and opposite reactions. PafA catalyzes a ligation reaction of a small protein tag to target protein substrates, whereas Dop removes the tag by hydrolysis of the iso-peptide bond between the tag and substrate. Given that both enzymes maintain a similar fold and high sequence homology, we wondered what are the critical differences in amino acid sequence and folding that are responsible for each distinct activity. We tackled this question using horizontal and vertical analysis of sequence-function relations, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity and yielded a catalytically active ligase. Our findings suggest that the change of activity is a result of a conformational change, with some conserved residues essential for stabilization of the Dop mutant. Most importantly, to our knowledge, this is one of a few examples that capture the emergence of a new catalytic function from a preexisting one.