Search Results (8)

Acetohydroxyacid synthase (AHAS) is a key enzyme in the first step of the branched-chain amino acid synthesis pathway, and the production of acetohydroxybutyrate from one molecule of 2-ketobutyric acid and one molecule of pyruvate. AHAS is inhibited by feedback from L-valine, L-leucine, and L-isoleucine, and the expression of ilvBN, the gene encoding AHAS, is regulated by all three branched-chain amino acids. A change in amino acids 20–22 on the regulatory subunit (M13 mutation) removes the feedback inhibition by valine. We cloned the gene encoding AHAS (ilvBN) into a vector and then transfected it into Escherichia coli BL21 for expression with targeted changes in amino acids 20–22 on the regulatory subunit, and then determined the activity of the mutated AHAS and its inhibitory effects on valine, isoleucine, and leucine. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Previous studies have suggested that the binding sites for the three branched-chain amino acids may be at the same variable center. We investigated the enzymatic properties of wild-type and mutant AHAS, modeled their crystal structures, and resolved the mechanism of feedback inhibition induced by mutant M13, which will be useful for continuing the modification of AHAS and the design of broad-spectrum herbicides. Full article

Herbicides are useful tools for managing weeds and promoting food production and sustainable agriculture. In this study, we report on the development of a novel class of lipophilic pyrimidine-biphenyl (PMB) herbicides. Firstly, three PMBs, Ia, IIa, and IIIa, were rationally designed via a scaffold hopping strategy and were determined to inhibit acetohydroxyacid synthase (AHAS). Computational simulation was carried out to investigate the molecular basis for the efficiency of PMBs against AHAS. With a rational binding mode, and the highest in vitro as well as in vivo potency, Ia was identified as a preferable hit. Furthermore, these integrated analyses guided the design of eighteen new PMBs, which were synthesized via a one-step Suzuki–Miyaura cross-coupling reaction. These new PMBs, Iba-ic, were more effective in post-emergence control of grass weeds compared with Ia. Interestingly, six of the PMBs displayed 98–100% inhibition in the control of grass weeds at 750 g ai/ha. Remarkably, Ica exhibited ≥ 80% control against grass weeds at 187.5 g ai/ha. Overall, our comprehensive and systematic investigation revealed that a structurally distinct class of lipophilic PMB herbicides, which pair excellent herbicidal activities with new interactions with AHAS, represent a noteworthy development in the pursuit of sustainable weed control solutions. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide health threat and has already tormented humanity during its long history, creating an urgent need for the development of new classes of antibacterial agents. In this study, twenty-one novel sulfonylurea derivatives containing phenyl-5-vinyl and pyrimidinyl-4-aryl moieties were designed and synthesized, among which, nine compounds exhibited inhibitory potencies against Gram-positive bacterial strains: MRSA (Chaoyang clinical isolates), S. aureus ATCC6538, vancomycin-resistant Enterococci-309 (VRE-309), and Bacillus subtilis ATCC 6633. Especially, 9i and 9q demonstrated inhibitory activities against the four bacterial strains with minimum inhibitory concentrations (MICs) of 0.78–1.56 μg/mL, and quite a few of other MRSA clinical strains with MICs of 0.78 μg/mL, superior to those of the positive controls vancomycin (MIC of 1 μg/mL) and methicillin (MIC of >200 μg/mL). This is the very first time that sulfonylurea derivatives have been identified as promising inhibitors against different MRSA clinical isolates. In addition, all the MIC values of the synthesized compounds against Candida albicans were greater than 100 μg/mL. Since the reported anti-Candida activities of sulfonylureas were due to acetohydroxyacid synthase (AHAS) inhibition, the molecular target against MRSA for the target sulfonylureas was thought to be a different mode of action. Density functional theory (DFT) calculations were finally performed to understand the structure–activity relationships, based on which, significant differences were observed between their HOMO maps for compounds with strong antibacterial activities and weak anti-MRSA effects. The present results hence provide valuable guidance for the discovery of novel agents to treat bacterial infections, especially against MRSA. Full article

Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicides but not to other groups of AHAS inhibitors, is described in the AHAS1 protein of EMS-mutagenized chickpea line M2033. Full article

Lolium rigidum Gaud. is a cross-pollinated species characterized by high genetic diversity and it was detected as one of the most herbicide resistance-prone weeds, globally. Acetohydroxyacid synthase (AHAS) resistant populations cause significant problems in cereal production; therefore, monitoring the development of AHAS resistance is widely recommended. Using next-generation sequencing (NGS), a de novo transcriptome sequencing dataset was presented to identify the complete open reading frame (ORF) of AHAS enzyme in L. rigidum and design markers to amplify fragments consisting of all of the eight resistance-conferring amino acid mutation sites. Pro197Thr, Pro197Ala, Pro197Ser, Pro197Gln, and Trp574Leu amino acid substitutions have been observed in samples. Although the Pro197Thr amino acid substitution was already described in SU and IMI resistant populations, this is the first report to reveal that the Pro197Thr in AHAS enzyme confers a high level of resistance (ED₅₀ 3.569) to pyroxsulam herbicide (Triazolopyrimidine). Full article

Background: The spread of herbicide-resistance Ambrosia artemisiifolia threatens not only the production of agricultural crops, but also the composition of weed communities. The reduction of their spread would positively affect the biodiversity and beneficial weed communities in the arable habitats. Detection of resistant populations would help to reduce herbicide exposure which may contribute to the development of sustainable agroecosystems. Methods: This study focuses on the application of target-site resistance (TSR) diagnostic of A. artemisiifolia caused by different herbicides. We used targeted amplicon sequencing (TAS) on Illumina Miseq platform to detect amino acid changes in herbicide target enzymes of resistant and wild-type plants. Results: 16 mutation points of four enzymes targeted by four herbicide groups, such as Photosystem II (PSII), Acetohydroxyacid synthase (AHAS), 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and protoporphyrinogen IX oxidase (PPO) inhibitors have been identified in common ragweed populations, so far. All the 16 mutation points were analyzed and identified. Out of these, two mutations were detected in resistant biotypes. Conclusions: The applied next-generation sequencing-targeted amplicon sequencing (NGS-TAS) method on A. artemisiifolia resistant and wild-type populations enable TSR detection of large sample numbers in a single reaction. The NGS-TAS provides information about the evolved herbicide resistance that supports the integrated weed control through the reduction of herbicide exposure which may preserve ecological properties in agroecosystems. Full article

The use of herbicides is an effective and economic way to control weeds, but their availability for rapeseed is limited due to the shortage of herbicide-resistant cultivars in China. The single-point mutation in the acetohydroxyacid synthase (AHAS) gene can lead to AHAS-inhibiting herbicide resistance. In this study, the inheritance and molecular characterization of the tribenuron-methyl (TBM)-resistant rapeseed (Brassica napus L.) mutant, K5, are performed. Results indicated that TBM-resistance of K5 was controlled by one dominant allele at a single nuclear gene locus. The novel substitution of cytosine with thymine at position 544 in BnAHAS1 was identified in K5, leading to the alteration of proline with serine at position 182 in BnAHAS1. The TBM-resistance of K5 was approximately 100 times that of its wild-type ZS9, and K5 also showed cross-resistance to bensufuron-methyl and monosulfuron-ester sodium. The BnAHAS1^544T transgenic Arabidopsis exhibited higher TBM-resistance than that of its wild-type, which confirmed that BnAHAS1^544T was responsible for the herbicide resistance of K5. Simultaneously, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS1^544T. In addition, a method for the fast screening of TBM-resistant plants at the cotyledon stage was developed. Our research identified and molecularly characterized one novel mutative AHAS allele in B. napus and laid a foundation for developing herbicide-resistant rapeseed cultivars. Full article

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice. Full article