David SchnettlerJan 3, 2023

Excited to share my very first paper which was just published in #JACS:

https://pubs.acs.org/doi/10.1021/jacs.2c10673

In this work carried out during my PhD together with Jamie Klein, Tomasz Kaminski, and Pierre-Yves Colin in the Hollfelder Lab at #UniversityofCambridge we evolve a #phosphotriesterase with a new, metal-independent mechanism by using droplet #microfluidics.

Comments and feedback welcome! A thread: (1/6)

#EnzymeEngineering #ProteinEngineering #EnzymeMechanism #DirectedEvolution #Bioremediation

Show threadDavid SchnettlerJan 3, 2023

Phosphotriesterases have evolved rapidly in the last decades in response to the large-scale pollution of environments with xenobiotic organophosphates (used for example as pesticides) – they could well be the youngest enzymes on the planet.

In an astonishing showcase of rapid convergent evolution, phosphotriesterases have emerged independently from a range of wildly different protein folds – remarkably all requiring a metal cofactor for efficient phosphotriester breakdown. (2/6)

Show threadDavid Schnettler

Here, we show that an alternative motif for phosphotriester hydrolysis exists, starting #DirectedEvolution from a metal-independent alpha/beta hydrolase which Pierre-Yves identified using functional #metagenomics:

https://www.nature.com/articles/ncomms10008

Phosphotriesters are so toxic because they irreversibly inhibit the catalytic triad of acetylcholinesterase. So, our starting point is a homologue of exactly this target, just with a cysteine instead of a serine in the catalytic triad. (3/6)

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics - Nature Communications

Environmental DNA from unculturable microorganisms contains genes with useful functions that remain difficult to identify and isolate. Here Colin, Kintses et al.demonstrate the screening of millions of samples in pL volumes to directly identify new enzymatic activities and complements sequence-based approaches.

Nature
Jan 3, 2023 at 8:58pmWeb
Show threadDavid SchnettlerJan 3, 2023

By combining smart libraries with the ultrahigh throughput of droplet #microfluidics, we increased its catalytic efficiency by 360-fold in only two rounds of evolution, matching the activity of most metal-dependent phosphotriesterases.

But how does this new triad-based phosphotriesterase escape the fate of acetylcholinesterase, irreversible covalent entrapment? (4/6)

Show threadDavid SchnettlerJan 3, 2023

By using steady and transient state #kinetics as well as linear free-energy relationships (#LFER) we show that the breakup of the covalent phosphoryl intermediate – which is the 'lethal' step for acetylcholinesterase – is surprisingly fast in this enzyme.

Instead, the rate-limiting step is the initial formation of the covalent intermediate. This step subsequently gets vastly accelerated through our directed evolution campaign (by increased leaving group stabilisation). (5/6)

Show threadDavid SchnettlerJan 3, 2023

Conclusions:

- Microfluidic droplet sorting is an extremely powerful tool for #proteinengineering
- Cys-triad enzymes might be predisposed and well suited for evolution towards pesticide decontamination or catalytic bioscavengers (6/6)