酮醇酸还原异构酶抑制剂的设计、合成及除草活性

酮醇酸还原异构酶(KARI)是一个有前景的除草剂靶标酶,有关其抑制剂的设计研究鲜有报道。在文献报道的菠菜KARI酶复合物0.165 nm高分辨率晶体结构基础上,利用分子对接DOCK 4.0方法进行MDL/ACD三维数据库搜寻,得到了279个与KARI酶结合能较低的小分子结构信息,并从中选取部分小分子进行化学合成,进而测试了其除草活性。在合成的17个化合物中,发现个别化合物对油菜 (抑制率70.8 %) 和稗草 (抑制率48.8 %)具有较好的生长抑制活性。     

乙酰乳酸合成酶及其抑制剂研究新进展

乙酰乳酸合成酶(AHAS)是支链氨基酸生物合成途径中的一个关键酶,是绿色除草剂的重要作用靶标。由于此生物合成过程只存在于植物和微生物体内,因此该类抑制剂对哺乳动物具有生物安全性。近年来,随着AHAS三维结构的阐明,人们不仅深入了解了已有抑制剂的作用机制,并且依此设计开发了一些新型的抑制剂,拓展了其在抑菌活性方面的生物学功能。文章对近年来AHAS及其抑制剂的最新研究进展进行了综述,重点就AHAS的酶学特征、结构特征及结合方式,以AHAS为靶标的新颖除草活性化合物的设计开发以及AHAS抑制剂的抗菌生物活性研究进展等问题详细进行了总结,以期为设计开发靶向AHAS的新型除草剂或抗菌药物提供参考。     

除草剂安全剂对作物细胞色素P450及其他酶活性和水平的影响

综述了除草剂安全剂对作物中参与除草剂解毒作用的酶以及作为除草剂作用靶标位点酶水平与活性的影响。安全剂能增强细胞色素P450酶系统活性,诱导P450在除草剂降解中的作用;增加作物体内谷胱甘肽的含量,从而促进除草剂与谷胱甘肽的轭合而发挥解毒作用;降低由于除草剂对乙酰乳酸合成酶的抑制作用而引起的植物毒性等。     

乙酰乳酸合成酶抑制剂的分子生物学和晶体学基础

乙酰乳酸合成酶（ALS）催化支链氨基酸生物合成中的第一步是包括磺酰脲类、咪唑啉酮类等多种除草剂的作用靶标。本文从基因学、分子生物学和晶体学的角度对ALS与ALS抑制剂的作用进行了综述,同时也阐述了ALS分子抗性机制。     

植物乙酰乳酸合成酶抑制剂作用方式及机理研究进展

以拟南芥Arabidopsis thaliana等植物为主要对象,系统评述了乙酰乳酸合成酶(ALS)抑制剂在作用靶标、选择性机制、毒性机理及化学杀雄作用等方面的研究进展。ALS是磺酰脲类、咪唑啉酮类等多种除草剂的共同作用靶标,最新研究又发现苯磺隆、酰嘧磺隆等多种ALS抑制剂可作为敏感植物的化学杀雄剂。目前该研究领域的薄弱环节是ALS抑制剂的毒性机理,先后提出了支链氨基酸饥饿、核酸合成受阻、ALS底物积累、养分转运障碍、无氧呼吸等假说,但均未能被证实。借助高通量的代谢组学、转录组学、蛋白质组学检测技术将能够更全面地揭示ALS抑制剂的生理生化效应,为研究其毒性机理提供新证据。     

新型化合物唑嘧氯草胺的作用机制

采用培养皿法和常规生化方法,研究了新型除草剂唑嘧氯草胺(暂定名,代号:ZJ-2725)的作用机制。结果显示,同时添加20mg/L浓度的缬氨酸、亮氨酸和异亮氨酸能完全恢复唑嘧氯草胺对苘麻芽的生长抑制作用,而添加相同浓度的单一支链氨基酸只能部分消除其抑制作用。离体条件下,随着唑嘧氯草胺浓度的增大,对乙酰乳酸合成酶(ALS)活性的抑制率增加,ALS活性随反应时间的延长而增加,且两者之间符合Michaelis-Meten方程;活体条件下,唑嘧氯草胺对苘麻的ALS也表现出明显的抑制作用,ALS活性明显下降。表明唑嘧氯草胺在植物体内抑制了3种支链氨基酸的生物合成,进而导致植物蛋白质合成受阻而使植物生长受到抑制,ALS即为唑嘧氯草胺的作用靶标。     

新型除草剂丙酯草醚的作用机理

采用常规的室内生测和生化方法,开展新型除草剂丙酯草醚的作用机理研究.试验结果表明:同时添加5mg/L浓度的三种支链氨基酸即缬氨酸、亮氨酸和异亮氨酸能完全恢复丙酯草醚对高粱茎的生长抑制作用,而添加相同浓度的单一支链氨基酸只能部分消除其抑制作用.离体条件下,丙酯草醚IC50＞100mg/L,而双草醚IC50值为10-5～10-4mg/L,说明丙酯草醚对离体ALS没有抑制作用;活体条件下,丙酯草醚对ALS有一定的抑制作用,且随着处理时间的延长,酶活力降低,对ALS的抑制作用增加.因此,丙酯草醚使植物体内必需的支链氨基酸合成受阻,仍然属于ALS抑制剂,但其作用方式不同于磺酰脲类和嘧啶水杨酸类除草剂等典型的ALS抑制剂,它类似于前体农药,即在植物体内通过代谢活化来发挥作用.     

农药中色素合成抑制剂的研究和应用进展

光合作用在植物的生长过程中起着至关重要的作用,以光合色素生物合成过程中的酶作为靶标,是研发除草剂的一个重要方向和热点。其中原卟啉原氧化酶(PPO),八氢番茄红素去饱和酶(PDS),ζ-胡萝卜素去饱和酶(ZDS),对羟苯基丙酮酸双氧化酶(HPPD)等作为除草剂靶酶非常成功。本文综述了近年来农药中色素合成抑制剂的作用机制及最新应用进展,并展望未来的发展趋势。     

EPSP合成酶的纯化与制备

EPSP合成酶 ( 5 -烯醇丙酮酸莽草酸 - 3-磷酸合酶 )是除草剂靶标酶之一 [1 ] ,也是抗草甘膦转基因作物的关键性酶 [2 ] ,它催化一分子的莽草酸 - 3-磷酸 ( S3P)和烯醇式丙酮酸 ( PEP)成EPSP,从而导致芳香族氨基酸——色氨酸、酪氨酸、苯丙氨酸的生物合成。 EPSP合成酶存在于细菌、真菌和植物中 ,但由于其性质的不稳定性、不均匀性和低丰度 ,造成分离纯化困难 [3] 。作者以菜豆 ( Phaselusvulgaris L .)幼苗为原料 ,经快速纯化 (整个纯化过程少于 1 .5 h)获得的EPSP合成酶产品能用于除草剂的筛选工作 ,为以 EPSP合成酶为靶标的生…     

靶标为原卟啉原氧化酶的高效除草剂品种

原卟啉原ＩＸ氧化酶是血红素和叶绿素生物合成中的关键酶，是过氧化型除草剂的分子靶标，当植物用二苯醚类，酞酰亚胺以及一些吡啶衍生物等除草剂处理后，造成原卟啉原ＩＸ积累，膜脂质破坏，最终细胞死亡，同ＡＬＳ抑制剂一样，作用靶标为原卟啉原化酶的除草剂，具有用药量低，活性高，杀草谱广，对哺乳动物低毒，对环境影响较小等良好特性，本文介绍了作用靶标为原卟啉原氧化酶的代表性品种。     

靶标酶在除草剂研究与开发中的应用

大多数除草剂都是通过特殊酶的抑制而产生杀草作用的。因此,以靶标进行分子设计,鉴定化合物分子结构中的活性团,开发能有效杀死杂草、而不伤害作物并对动物及环境安全的除草剂品种有着重要意义。本文着重对各种不同类型靶标酶在除草剂的研究与开发过程中的应用加以阐述。     

靶标酶在除草剂研究与开发中的作用

大多数除草剂都是通过特殊酶的抑制而产生杀草作用的。根据作用靶标对除草剂进行分类,了解靶标酶的作用机理及特性,对于新型除草剂的设计和杂草抗性的防治能够起到很大的帮助。本文介绍了谷胱甘肽转移酶(GST)、细胞色素P450酶、乙酰乳酸合成酶(ALS)、乙酞辅酶A羧化酶(ACCace)的研究进展。并分别从酶的生理功能、酶学特征、抑制剂作用机理、抑制剂的研究、抗性等方面进行了不同程度的阐述。     

Moth sex-pheromone biosynthesis is inhibited by the herbicide diclofop

Pheromones of nocturnal moths are derived from fatty acids produced as a result of the activity of acetyl-CoA carboxylase. This timely production is initiated in nocturnal moths by a tropic peptide, pheromone biosynthesis activating neuropeptide released into the hemolymph. In monocotyledonous plants, specific plastid acetyl-CoA carboxylase is inhibited by herbicides that target the eukaryotic form of the enzyme. We report evidence that these herbicides can also target pheromone biosynthesis by a moth, thereby implicating the acetyl-CoA carboxylase as a key regulatory enzyme in the pheromone biosynthetic pathway. These findings, whilst indicating the possible action of such herbicides on non-target organisms, also suggest a novel alternative method of insect pest management, which precludes sex-pheromone production and mating success, thereby reducing insect population growth.     

Quinone activation of protoporphyrinogen oxidase of barley plastids

Protoporphyrinogen oxidase catalyzing the oxidation of protoporphyrinogen to protoporphyrin is the target enzyme for several highly active herbicides. The plastidic plant enzyme under normal conditions uses oxygen as electron acceptor. Duroquinone, however, can be an alternative electron acceptor of protoporphyrinogen oxidase of barley plastids. In this respect the enzyme from the plastids may be an evolutionary intermediate between bacterial enzymes coupled to ubiquinone and mammalian mitochondrial enzymes coupled to oxygen.     

Interaction between acetylcholinesterase and choline acetyltransferase: an hypothesis to explain unusual toxicological responses

Organophosphorus and carbamate insecticides are thought to have only one target site, acetylcholinesterase (EC 3.1.1.7). When this enzyme is inhibited, the neurotransmitter acetylcholine is not metabolized and polarization of the post-synaptic membrane does not take place. But, what happens when the cholinesterase becomes resistant or when neurotransmitter levels are diminished? Here, we report results suggesting that choline acetyltransferase (EC 2.3.1.6), the enzyme responsible for the acetylcholine production, may be involved either as an alternative pesticide target site or as a factor enhancing survival during insecticide exposure. This underlines the concept that the pivotal step for insecticide toxicology is not the acetylcholinesterase activity but the amount of acetylcholine present. This latter can only fluctuate between an upper and a lower threshold, and crossing one of these two thresholds leads to the death of the insect. The interaction between acetylcholinesterase and choline acetyltransferase activities would explain the astonishing toxicological phenomenon that, in some conditions, mortality decreases when insecticide concentration increases. ©1997 SCI     

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.     

A novel genomic approach to herbicide and herbicide mode of action discovery

A herbicide with a new mode of action has not been commercialized for more than 30 years. A recent paper describes a novel genomic approach to herbicide and herbicide mode of action discovery. Analysis of a microbial gene cluster revealed that it encodes genes for both the biosynthetic pathway for production of the sesquiterpene aspterric acid and an aspterric acid‐resistant form of dihydroxy acid dehydratase (DHAD), its target enzyme. Aspterric acid is weak compared with commercial synthetic herbicides, and whether DHAD is a good herbicide target is unclear from this study. Nevertheless, this genomic approach provides a novel strategy for the discovery of herbicides with new modes action. © 2018 Society of Chemical Industry     

Resistance to glyphosate in Lolium rigidum

Annual ryegrass (Lolium rigidum) is a widespread and important weed of Australia and populations of this weed have developed resistance to most major herbicides, including glyphosate. The possible mechanisms of resistance have been examined in one glyphosate-resistant Lolium population. No major differences were observed between resistant and susceptible biotypes in respect of (i) the target enzyme (EPSP synthase), (ii) DAHP synthase, the first enzyme of the target (shikimate) pathway, (iii) absorption of glyphosate, or (iv) translocation. Following treatment with glyphosate, there was greater accumulation of shikimate (derived from shikimate-3-Pi) in susceptible than in resistant plants. In addition, the resistant population exhibited cross-resistance to 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate, a herbicide which, although structurally similar to glyphosate, acts at an unrelated target site. On the basis of these observations we speculate that movement of glyphosate to its site of action in the plastid is involved in the resistance mechanism. © 1999 Society of Chemical Industry