posted on 2022-12-30, 13:05authored byJ. David Schnettler, Oskar James Klein, Tomasz S. Kaminski, Pierre-Yves Colin, Florian Hollfelder
Finding new mechanistic solutions for biocatalytic challenges
is
key in the evolutionary adaptation of enzymes, as well as in devising
new catalysts. The recent release of man-made substances into the
environment provides a dynamic testing ground for observing biocatalytic
innovation at play. Phosphate triesters, used as pesticides, have
only recently been introduced into the environment, where they have
no natural counterpart. Enzymes have rapidly evolved to hydrolyze
phosphate triesters in response to this challenge, converging onto
the same mechanistic solution, which requires bivalent cations as
a cofactor for catalysis. In contrast, the previously identified metagenomic
promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves
slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp
triad. Here, we probe the evolvability of this new catalytic motif
by subjecting P91 to directed evolution. By combining a focused library
approach with the ultrahigh throughput of droplet microfluidics, we
increase P91’s activity by a factor of ≈360 (to a kcat/KM of ≈7
× 105 M–1 s–1)
in only two rounds of evolution, rivaling the catalytic efficiencies
of naturally evolved, metal-dependent phosphotriesterases. Unlike
its homologue acetylcholinesterase, P91 does not suffer suicide inhibition;
instead, fast dephosphorylation rates make the formation of the covalent
adduct rather than its hydrolysis rate-limiting. This step is improved
by directed evolution, with intermediate formation accelerated by
2 orders of magnitude. Combining focused, combinatorial libraries
with the ultrahigh throughput of droplet microfluidics can be leveraged
to identify and enhance mechanistic strategies that have not reached
high efficiency in nature, resulting in alternative reagents with
novel catalytic machineries.