Engineering enzymes with enhanced activity and stability is a central goal of biotechnology, yet the inherent trade-off between optimizing global protein fitness and specific substrate binding affinity poses a significant challenge. Here, we present ESM-FEP, a computational framework that synergistically integrates a fine-tuned protein language model with alchemical free energy perturbation (FEP) to overcome this limitation. Our workflow employs a parameter-efficient fine-tuned ESM-2 model to perform high-throughput saturation mutagenesis, rapidly identifying mutations that preserve protein fitness. Top-ranking candidates are then subjected to rigorous FEP simulations to precisely quantify changes in substrate binding affinity. When applied to engineer the Zea mays dioxygenase ZmHSL1B for improved detoxification of the herbicide mesotrione, ESM-FEP efficiently navigated the mutational landscape and identified a quadruple mutant M5 (Q140H/Y205F/L332R/K336F). This variant demonstrated a catalytic efficiency approximately 7-fold higher than that of the wild-type enzyme, which was corroborated by in vitro assays and a detailed kinetic analysis. Furthermore, transgenic Arabidopsis thaliana expressing the engineered mutant M5 exhibited significantly enhanced herbicide tolerance, validating its functional efficacy in a biological context. The ESM-FEP framework establishes a generalizable and efficient strategy for the rational design of gain-of-function enzymes, with broad applications in biocatalysis, bioremediation, and precision agriculture.
Dear Editor , The detoxi fi cation of phytotoxic compounds is a prerequisite for plant survival. ATP-binding cassette family C (ABCC) transporters play a pivotal role in the export of toxic compounds into vacuoles, a critical step in detoxi fi cation 1 . Arabidopsis thaliana ABCC2 ( At ABCC2) is responsible for the ef fl ux of glutathione conjugates of pesticides, such as atrazine and metolachlor, into the vacuoles 2 – 5 . Additionally, At ABCC2 can export arsenic-phytochelatin conjugates into vacuoles, resulting in increased arsenic tolerance 6,7 . Despite its critical role in plant detoxi fi cation, the biochemical and structural mechanisms underlying the function of At ABCC2 remain incompletely understood. To address this gap, we present cryo-electron microscopy (cryo-EM) structures of At ABCC2 in four states: apo, substrate bound, closed, and dimeric. Structural analysis revealed a unique architecture, distinguished by the atypical localization of its transmembrane domain 0 (TMD0) domain. Moreover, biochemical studies revealed that the TMD0 domain is critical for coordinating transport channel closure. The molecular basis of atrazine export by At ABCC2 was also determined. Additionally, the plant-speci fi c dimerization of At ABCC2 was demonstrated to be mediated by TMD2 and nucleotide-binding domain 2 (NBD2) rather than by the TMD0 domain. Notably, we found that although dimeric At ABCC2 represented a physiological form, its dimerization resulted in reduced substrate export activity. These fi ndings provide new insights into the detoxi fi cation mechanism of At ABCC2 and highlight the potential for using ABCC transporters to develop herbicide-resistant crops. Full-length At
Pyridalyl is a novel insecticide with a unique mode of action, but resistance in Plutella xylostella is a significant challenge. To elucidate the resistance mechanism, we compared transcriptomes of susceptible (XY-PS) and laboratory-selected resistant (XY-PR) strains. The mitochondrial cytochrome P450 gene CYP12A2 emerged as a key candidate. RT-qPCR confirmed CYP12A2 was upregulated approximately 8-fold in XY-PR and 9-fold in a field-collected resistant strain (ZL-PR). Furthermore, RNA interference (RNAi) targeting CYP12A2 significantly increased larval susceptibility to pyridalyl. Recombinant CYP12A2 expressed in Sf9 cells specifically metabolized pyridalyl, producing a distinct hydroxylated metabolite identified via UPLC-MS/MS. These findings represent direct evidence of P450-mediated metabolic detoxification of pyridalyl. Ultimately, this unveils a novel biochemical adaptation in P. xylostella, establishing CYP12A2 as a critical biomarker for field resistance monitoring and a promising target for integrated insecticide resistance management (IRM) strategies.
Falong Wang, Chunyan Yin, Zhiyuan Qin et al.· Journal of Agricultural and...· 0 citations
4-Hydroxyphenylpyruvate dioxygenase (HPPD) has become an attractive target for herbicide development owing to the slow development and low risk of resistance to its inhibitors. However, most available HPPD inhibitors exhibit limited crop selectivity and poor efficacy against grasses. Therefore, the exploration of novel molecular scaffolds is of great research value. In this study, we adopted a ring-fusion strategy to enhance π-π stacking interactions and developed a structurally novel pyrazole-4-chloro-benzimidazolin-2-one scaffold. Through systematic structural optimization, compound II-19 showed the strongest enzyme inhibitory activity, with an IC50 of 52 nM, about 6-fold higher potency than the positive control, mesotrione. The crystal structure of AtHPPD-II-19 revealed that the inhibitor engages in characteristic chelation and π-π stacking interactions within the active site, and the 3-NO2-benzyl group contributes additional hydrophobic interactions and a possible weak water-mediated interaction involving Gln293. Additionally, I-5 showed excellent broad-spectrum weed control and good safety toward peanut at 30 g a.i./ha. These results indicate that compound I-5 is a promising candidate for the development of new HPPD inhibitors for weed control in peanut fields.
Biao Li, Rui-Ning Ying, Xian-Quan Wang et al.· Journal of Agricultural and...· 0 citations