Schnettler Fernandez, D. | Search bioRxiv

bioRxiv2023

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function required a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain and introduced a catalytically important cysteine. The truncated protein dimerized into a dynamic -helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration on the order of 109 and catalytic proficiency on the order of 1014 M-1, comparable to large enzymes shaped by billions of years of evolution. These findings suggest that fragmentation to modular primordial peptides can be a fertile avenue for introducing structural and functional diversity into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/528392v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@180e004org.highwire.dtl.DTLVardef@1f32a75org.highwire.dtl.DTLVardef@16876aforg.highwire.dtl.DTLVardef@147ffc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase

The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators. Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the /{beta}-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105 members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91s phosphotriesterase activity was increased {approx} 400-fold to a kcat/KM of {approx} 106 M-1s-1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, 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 a novel catalytic machinery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/480337v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@5e6f7aorg.highwire.dtl.DTLVardef@1e8cd72org.highwire.dtl.DTLVardef@10809c9org.highwire.dtl.DTLVardef@ba6fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗