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

TL;DR

Scientists used directed evolution to convert an enzyme that breaks down isochorismate into a more efficient enzyme that transforms chorismate.

Contribution

The study demonstrates how directed evolution can reverse enzyme function and uncovers a potential evolutionary link between two enzyme types.

Findings

A PchB variant with 10-fold increased chorismate mutase activity was evolved.

A final PchB variant achieved a k_cat/K_m of 96,000 M–1 s–1, comparable to natural chorismate mutases.

Existing chorismate mutases were found to have unexpected isochorismate pyruvate lyase activity.

Abstract

PchB is an isochorismate pyruvate lyase (IPL) involved
in siderophore
biosynthesis in . 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 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
cat/K
m of 96,000
M–1 s–1, 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 . 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.

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