Biology subjects

Carr, P. D.

Publications and source records attributed to Carr, P. D..

4 recordsLinked to original sources

bioRxiv2025

Structural and Evolutionary Constraints of Organophosphate Resistance in Dipteran Carboxylesterases

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G[->]D mutation in the oxyanion hole of -carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyse structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1000-fold vs. the wild-type enzyme and tested these variants in transgenic Drosophila melanogaster, demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others. SignificanceThis study reveals the intricate evolutionary path to insecticide resistance in insects, highlighting why a potent resistance mutation is effective in some species but not others. We show that the mutations success is contingent on the enzymes pre-existing structural features, highlighting the strong intramolecular epistasis. Using laboratory evolution, we enhanced the enzymes detoxification activity over 1000-fold, yet discovered this did not translate to increased resistance in transgenic flies. This surprising result demonstrates that effective real-world resistance requires a delicate balance between an enzymes ability to bind an insecticide (affinity) and its speed at breaking it down (turnover), providing crucial insights into the constraints governing molecular adaptation.

biochemistry↗

bioRxiv2018

Higher-order epistatic networks underlie the evolutionary fitness landscape of a xenobiotic- degrading enzyme

Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural, and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), a xenobiotic organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that a complex interaction network, defined in part by higher-order epistasis, determined the adaptive pathways that were available. By also characterizing the adaptive landscapes in terms of their functional activity towards three other OP substrates, we reveal that subtle differences in substrate substituents drastically alter the enzymes epistatic network by changing its intramolecular interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.

biochemistry↗

bioRxiv2017

Cryptic genetic variation defines the adaptive evolutionary potential of enzymes

Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may also impact the evolutionary potential of proteins, but the molecular basis for this remains unclear. Here we perform comparative directed evolution in which four orthologous metallo-{beta}-lactamases were evolved toward a new function. We found that genetic variation between these enzymes resulted in distinct evolutionary outcomes. The ortholog with the lower initial activity reached a 20-fold higher fitness plateau exclusively via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show that the cryptic molecular properties and conformational variation of residues in the initial genotypes cause epistasis, thereby constraining evolutionary outcomes. Our work highlights that understanding the molecular details relating genetic variation to protein functions is essential to predicting the evolution of proteins.

evolutionary biology↗

bioRxiv2017

Evolution of an enzyme from a solute-binding protein

Much of the functional diversity observed in modern enzyme superfamilies originates from molecular tinkering with existing enzymes1. New enzymes frequently evolve from enzymes with latent, promiscuous activities2, and often inherit key features of the ancestral enzyme, retaining conserved catalytic groups and stabilizing analogous intermediates or transition states3. While experimental evolutionary biochemistry has yielded considerable insight into the evolution of new enzymes from existing enzymes4, the emergence of catalytic activity de novo remains poorly understood. Although certain enzymes are thought to have evolved from non-catalytic proteins5-7, the mechanisms underlying these complete evolutionary transitions have not been described. Here we show how the enzyme cyclohexadienyl dehydratase (CDT) evolved from a cationic amino acid-binding protein belonging to the solute-binding protein (SBP) superfamily. Analysis of the evolutionary trajectory between reconstructed ancestors and extant proteins showed that the emergence and optimization of catalytic activity involved several distinct processes. The emergence of CDT activity was potentiated by the incorporation of a desolvated general acid into the ancestral binding site, which provided an intrinsically reactive catalytic motif, and reshaping of the ancestral binding site, which facilitated enzyme-substrate complementarity. Catalytic activity was subsequently gained via the introduction of hydrogen-bonding networks that positioned the catalytic residue precisely and contributed to transition state stabilization. Finally, catalytic activity was enhanced by remote substitutions that refined the active site structure and reduced sampling of non-catalytic states. Our work shows that the evolutionary processes that underlie the emergence of enzymes by natural selection in the wild are mirrored by recent examples of computational design and directed evolution of enzymes in the laboratory.

biochemistry↗