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

Publication date: Jul 22, 2025

PchB is an isochorismate pyruvate lyase (IPL) involved in siderophore biosynthesis in Pseudomonas aeruginosa. Besides catalyzing the [1,5]-sigmatropic rearrangement of isochorismate, PchB also has weak chorismate mutase (CM) activity, promoting the [3,3]-sigmatropic transformation of chorismate. It has been suggested that the secondary metabolism enzyme PchB evolved from a primary metabolism CM precursor. Here, we employed directed evolution to convert PchB (back) into an efficient CM. A total of seven residues around the active site differing between PchB and a prototypical CM from Escherichia coli were randomized, and the resulting gene library was subjected to selection for CM activity. After growth selection in an auxotrophic strain, a catalyst with 10-fold increased CM activity emerged. The improved enzyme was again randomized at three active site positions and subjected to selection, leading to a PchB variant with a k/K of 96,000 M s, which is 40 times higher than that of the parent enzyme and well within the range of dedicated natural CMs. The facile conversion of an IPL into a CM by directed evolution coincides with the fact that both reactions proceed through mechanistically interesting pericyclic processes, reaction types otherwise rarely used by enzymes. When probing typical established CMs for catalytic promiscuity, we discovered spurious IPL activity for the secreted CM from Mycobacterium tuberculosis. Our results hint at active site features, particularly a Val at the bottom of the substrate-binding pocket that may have served as a steppingstone for the evolution of IPL activity in a primordial CM.

Semantics

Original Article