Exploring Protein Space: From Hydrolase to Ligase by Substitution.

Area of Science:

• Protein evolution and enzyme function
• Biochemistry and structural biology

Background:

• Understanding how proteins evolve new functions is a key biological question.
• Two homologous bacterial enzymes, PafA (ligase) and Dop (hydrolase), perform distinct but related functions on protein tags.
• These enzymes share similar folds and sequence homology, making them ideal for studying functional divergence.

Purpose of the Study:

• To identify the specific amino acid sequence and structural differences responsible for the distinct activities of PafA and Dop.
• To elucidate the molecular mechanisms underlying the evolution of novel catalytic functions from existing ones.

Main Methods:

• Analysis of sequence-function relationships between PafA and Dop.
• Site-directed mutagenesis, specifically reciprocal mutagenesis of the Dop enzyme.
• Analysis of crystal structures of Dop and PafA to understand conformational changes.

Main Results:

• Identification of uniquely conserved residues in each enzyme critical for their specific activities.
• Reciprocal mutagenesis of Dop abolished its hydrolase activity and created a ligase activity.
• A conformational change in a key active site loop, stabilized by conserved residues, underlies the activity switch.

Conclusions:

• Specific residues and structural elements dictate the distinct catalytic functions of PafA and Dop.
• The study reveals the molecular changes required for the emergence of new enzymatic functions from ancestral ones.
• Evolutionary pathways leading to functional divergence can involve conserved positions and alternative mutational routes.