Evolutionary Specialization of a Promiscuous Designer Enzyme

The evolution of
a promiscuous enzyme for its various activities
often results in catalytically specialized variants. This is an important
natural mechanism to ensure the proper functioning of natural metabolic
networks. It also acts as both a curse and blessing for enzyme engineers,
where enzymes that have undergone directed evolution may exhibit exquisite
selectivity at the expense of a diminished overall catalytic repertoire.
We previously performed two independent directed evolution campaigns
on a promiscuous designer enzyme that leverages the unique properties
of a noncanonical amino acid (ncAA) para-aminophenylalanine
(pAF) as catalytic residue, resulting in two evolved variants which
are both catalytically specialized. Here, we combine mutagenesis,
crystallography, and computation to reveal the molecular basis of
the specialization phenomenon. In one evolved variant, an unexpected
change in quaternary structure biases substrate dynamics to promote
enantioselective catalysis, while the other demonstrates synergistic
cooperation between natural side chains and the pAF residue to form
semisynthetic catalytic machinery.