Exploring Protein Space: From Hydrolase to Ligase by Substitution

Nir Hecht, Caroline L. Monteil, Guy Perrière, Marina Vishkautzan, Eyal Gur

Research output: Contribution to journalArticlepeer-review


The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

Original languageAmerican English
Pages (from-to)761-776
Number of pages16
JournalMolecular Biology and Evolution
Issue number3
StatePublished - 1 Mar 2021


  • Dop
  • PafA
  • Pup
  • molecular evolution
  • structure-function

All Science Journal Classification (ASJC) codes

  • Ecology, Evolution, Behavior and Systematics
  • Molecular Biology
  • Genetics


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