(Reverse) Evolution of a Promiscuous Isochorismate Pyruvate Lyase into an Efficient Chorismate Mutase.

Area of Science:

• Enzyme Engineering and Directed Evolution
• Biochemical Mechanisms and Enzymology
• Evolutionary Biochemistry

Background:

• PchB, an isochorismate pyruvate lyase (IPL) in *Pseudomonas aeruginosa*, possesses weak chorismate mutase (CM) activity.
• It is hypothesized that PchB evolved from a primary metabolism CM precursor, suggesting a potential evolutionary link.
• Both IPL and CM activities involve mechanistically complex pericyclic reactions, which are rare in enzymatic catalysis.

Purpose of the Study:

• To engineer PchB, an IPL, into an efficient chorismate mutase (CM) using directed evolution.
• To investigate the evolutionary relationship between IPL and CM enzymes.
• To explore the catalytic promiscuity of known CMs and identify potential evolutionary pathways.

Main Methods:

• Directed evolution was employed to enhance the CM activity of PchB.
• Mutagenesis of active site residues, followed by selection for improved CM activity in an auxotrophic strain.
• Characterization of enzyme kinetics and screening for promiscuous activities in natural CMs.

Main Results:

• A PchB variant with a 10-fold increase in CM activity was obtained after the first round of directed evolution.
• A subsequent round of evolution yielded a PchB variant with a 40-fold higher catalytic efficiency (kcat/Km), comparable to natural CMs.
• The secreted CM from *Mycobacterium tuberculosis* exhibited spurious IPL activity, suggesting a potential evolutionary precursor role.

Conclusions:

• Directed evolution can efficiently convert an isochorismate pyruvate lyase into a highly active chorismate mutase.
• The study supports the hypothesis of evolutionary plasticity between IPL and CM enzymes, possibly involving a primordial CM.
• Specific active site features, like a Val residue, may facilitate the evolution of novel enzymatic activities such as IPL.