Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase.

Homogentisate solanesyltransferase (HST) is a crucial enzyme in the plastoquinone biosynthetic pathway and has recently emerged as a promising target for herbicides. In this study, we successfully expressed and purified a stable and highly pure form of seven times transmembrane protein Chlamydomonas reinhardtii HST (CrHST). The final yield of CrHST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on CrHST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC50 value to be 3.63 ± 0.53 µM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of CrHST with two substrates, determining the Km values as 22.76 ± 1.70 µM for FPP and 48.54 ± 3.89 µM for HGA. Through sequence alignment analyses and three-dimensional structure predictions, we identified conserved amino acid residues forming the active cavity in the enzyme. The results from molecular docking and binding energy calculations indicate that DMC has a greater binding affinity with HST compared to HGA. These findings represent substantial progress in understanding CrHST's properties and potential for herbicide development. KEY POINTS: • First high-yield transmembrane CrHST protein via E. coli system • Preliminarily identified active cavity composition via activity testing • Determined substrate and inhibitor modes via molecular docking.

Discovery of Subnanomolar Inhibitors of 4-Hydroxyphenylpyruvate Dioxygenase via Structure-Based Rational Design.

High-potency 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are usually featured by time-dependent inhibition. However, the molecular mechanism underlying time-dependent inhibition by HPPD inhibitors has not been fully elucidated. Here, based on the determination of the HPPD binding mode of natural products, the π-π sandwich stacking interaction was found to be a critical element determining time-dependent inhibition. This result implied that, for the time-dependent inhibitors, strengthening the π-π sandwich stacking interaction might improve their inhibitory efficacy. Consequently, modification with one methyl group on the bicyclic ring of quinazolindione inhibitors was achieved, thereby strengthening the stacking interaction and significantly improving the inhibitory efficacy. Further introduction of bulkier hydrophobic substituents with higher flexibility resulted in a series of HPPD inhibitors with outstanding subnanomolar potency. Exploration of the time-dependent inhibition mechanism and molecular design based on the exploration results are very successful cases of structure-based rational design and provide a guiding reference for future development of HPPD inhibitors.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.