Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β- Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase

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 CysHis-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 approximate to 360 (to a kcat/KM of approximate to 7 x 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.

Automated summary

Finding new mechanistic solutions for biocatalytic challenges is crucial in the evolution of enzymes and the development of new catalysts. Recent introduction of man-made substances into the environment offers an opportunity to observe biocatalytic innovation. Enzymes have rapidly adapted to hydrolyze phosphate triesters, converging onto a common mechanistic solution requiring bivalent cations. In contrast, the enzyme P91 achieves slow phosphotriester hydrolysis through a metal-independent triad, but its catalytic efficiency is improved by directed evolution.