17 research outputs found
Laboratory-directed evolution as a tool for anticipating insecticide resistance
The evolution of insecticide resistance provides a eukaryotic model system for studying enzyme evolution. Understanding the molecular basis of insecticide resistance can assist both the development of new methods to combat resistance and the anticipation of future resistance. Three insect species have independently evolved catalytic organophosphate (OP) insecticide resistance through a single active-site mutation (Gly\u3eAsp) in the αE7 enzyme1-3. To explore the evolutionary potential of αE7, we subjected αE7 from the blowfly Lucilia cuprina to nine rounds of mutation and selection, resulting in a \u3e1000-fold increase in OP-hydrolase activity and a kcat / KM \u3e 106 M-1 min-1. Kinetic and structural analysis of the evolutionary trajectory revealed the molecular basis for the increase in catalytic efficiency. Mutations occurring in the early stages of the trajectory enrich the productive side chain conformation of the key aspartic acid residue, while mutations in later stages remodel the binding pocket. Remarkably, mutations appearing in the later rounds yielded larger improvements in catalytic efficiency compared to initial mutations, indicating that the initial Gly\u3eAsp mutation represents only a fraction of the αE7 evolutionary potential. Worryingly, this suggests that the Gly\u3eAsp could be the first of many steps toward efficient OP-insecticide detoxification.
Please click Additional Files below to see the full abstract
The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature.
Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency
The evolution of new enzymatic activity
is rarely observed outside
of the laboratory. In the agricultural pest <i>Lucilia cuprina</i>, a naturally occurring mutation (Gly137Asp) in α<i>-</i>esterase 7 (<i>Lc</i>αE7) results in acquisition
of organophosphate hydrolase activity and confers resistance to organophosphate
insecticides. Here, we present an X-ray crystal structure of <i>Lc</i>αE7:Gly137Asp that, along with kinetic data, suggests
that Asp137 acts as a general base in the new catalytic mechanism.
Unexpectedly, the conformation of Asp137 observed in the crystal structure
obstructs the active site and is not catalytically productive. Molecular
dynamics simulations reveal that alternative, catalytically competent
conformers of Asp137 are sampled on the nanosecond time scale, although
these states are less populated. Thus, although the mutation introduces
the new reactive group responsible for organophosphate detoxification,
the catalytic efficiency appears to be limited by conformational disorganization:
the frequent sampling of low-energy nonproductive states. This result
is consistent with a model of molecular evolution in which initial
function-changing mutations can result in enzymes that display only
a fraction of their catalytic potential due to conformational disorganization
Overcoming insecticide resistance through computational inhibitor design
Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy
Of problems and opportunities—How to treat and how to not treat crystallographic fragment screening data
In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist
Recommended from our members
Of problems and opportunities-How to treat and how to not treat crystallographic fragment screening data.
In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist
Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila
International audiencePrevious electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate
Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability
Recommended from our members
Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation