Phytodetoxification of the explosive 2,4,6-trinitrotoluene

爆炸物 2,4,6-三硝基甲苯的植物解毒

• 批准号: BB/P005713/1
• 负责人: Neil Bruce
• 金额: $ 63.81万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP005713%2F1
• 关键词: Phytodetoxification; explosive; trinitrotoluene

The explosive 2,4,6-trinitrotoluene (TNT) has become an extensive global pollutant over the last 100 years and there are mounting concerns over the toxicity of TNT to biological systems. During World War I and II the toxic effects of TNT were discovered during large scale production, with 475 fatalities and over 17,000 TNT poisoning cases reported at manufacturing facilities. TNT has been shown to severely impact the diversity of soil microbial communities and the establishment of vegetation. In the U.S. alone it is estimated that some 10 million hectares of military land is contaminated with munitions constituents. Unlike similar situations where the environment has become contaminated with toxic agrochemicals and their use subsequently banned, the huge demand for military explosives means that TNT will continue to be manufactured and used globally on a massive scale for the foreseeable future.Because of the scale of explosives pollution, particularly on military training ranges, there is considerable interest in developing plant based remediation strategies. Plants offer a low cost sustainable solution to containing and remediating explosives pollution. However, a fundamental understanding of the phytotoxicity of explosives, and the enzyme systems plants use to detoxify these compounds, and the rate-limiting steps, are required to enable the development of robust plant systems to contain and remediate explosives pollution effectively in situ. In plants, the majority of TNT remains in the roots, where it inhibits growth and development reducing whole plant biomass. We have recently discovered that the mitochondria- and plastid-targeted enzyme monodehydroascorbate reductase 6 (MDHAR6) reduces TNT by one electron, forming a nitro radical which reacts with atmospheric oxygen, generating highly reactive superoxide. This futile catalytic cycle only requires catalytic quantities of TNT to continuously generate damaging reactive oxygen species in the mitochondria. We have demonstrated that mutants in MDHAR6 have dramatically enhanced TNT tolerance, and we propose that this reaction accounts almost entirely for TNT toxicity in plants. The major goal of this research programme is to rigorously and quantitatively establish the fate and effects of TNT on plants. In order to achieve this objective we propose to study mechanisms of TNT toxicity and fully elucidate TNT induced detoxification pathways that include glucosylation, glutathionylation and oxidative activity by cytochromes P450. The fate of the TNT metabolites produced by these enzymes will then be established. We have previously demonstrated that the TNT active glutathione transferase GST-U25 results in the removal of a nitro group which could render the aromatic ring more amenable to biodegradation. We now have detailed structural information on GST-U25 that will allow us to engineer and improve the specificity and activity towards TNT. We hope to use the knowledge gained from this study to develop improved plant systems that will clean up polluted sites and prevent explosives pollution from contaminating water sources.

过去 100 年来，爆炸性 2,4,6-三硝基甲苯 (TNT) 已成为一种广泛的全球污染物，人们越来越担心 TNT 对生物系统的毒性。第一次世界大战和第二次世界大战期间，TNT 的毒性作用在大规模生产过程中被发现，制造工厂报告了 475 人死亡和超过 17,000 起 TNT 中毒案例。 TNT 已被证明会严重影响土壤微生物群落的多样性和植被的建立。据估计，仅在美国就有约 1000 万公顷的军事用地受到弹药成分的污染。与环境受到有毒农用化学品污染并随后被禁止使用的类似情况不同，对军用炸药的巨大需求意味着在可预见的未来，TNT将继续在全球范围内大规模生产和使用。由于炸药的规模污染，特别是军事训练场的污染，人们对开发基于植物的修复策略非常感兴趣。工厂为控制和修复爆炸物污染提供了一种低成本的可持续解决方案。然而，需要对爆炸物的植物毒性、植物用于解毒这些化合物的酶系统以及限速步骤有基本的了解，才能开发出强大的植物系统，以有效地就地遏制和修复爆炸物污染。在植物中，大部分 TNT 残留在根部，抑制生长和发育，减少整个植物的生物量。我们最近发现，针对线粒体和质体的单脱氢抗坏血酸还原酶 6 (MDHAR6) 可通过一个电子还原 TNT，形成硝基，与大气中的氧气发生反应，生成高活性超氧化物。这种徒劳的催化循环只需要催化量的 TNT 即可在线粒体中持续产生破坏性的活性氧。我们已经证明MDHAR6的突变体显着增强了TNT耐受性，并且我们认为这种反应几乎完全解释了植物中的TNT毒性。该研究项目的主要目标是严格定量地确定 TNT 对植物的命运和影响。为了实现这一目标，我们建议研究 TNT 毒性机制，并充分阐明 TNT 诱导的解毒途径，包括细胞色素 P450 的糖基化、谷胱甘肽化和氧化活性。由这些酶产生的 TNT 代谢物的命运随后将被确定。我们之前已经证明，TNT 活性谷胱甘肽转移酶 GST-U25 可以去除硝基，从而使芳环更容易生物降解。我们现在拥有 GST-U25 的详细结构信息，这将使我们能够设计和改进 TNT 的特异性和活性。我们希望利用从这项研究中获得的知识来开发改进的工厂系统，以清理污染场地并防止爆炸物污染水源。

项目成果

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

辅因子消耗筛选确定了有前景的 NfsB 家族硝基还原酶用于二硝基甲苯修复。

• DOI: 10.1007/s10529-019-02716-z
• 发表时间: 2019
• 期刊: Biotechnology letters
• 影响因子: 2.7
• 作者: Williams EM