Phytodetoxification of the explosive 2,4,6-trinitrotoluene

爆炸性的2,4,6-三硝基甲苯（TNT）在过去的100年里已经成为一种广泛的全球性污染物，人们越来越关注TNT对生物系统的毒性。在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中被发现，有475人死亡，超过17,000例TNT中毒病例报告在生产设施。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，仅在美国就有大约1000万公顷的军用土地被军火成分污染。与环境被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。由于爆炸物污染的规模，特别是在军事训练场，有相当大的兴趣发展基于植物的补救策略。工厂为控制和修复炸药污染提供了一种低成本的可持续解决方案。然而，对爆炸物的植物毒性、植物用来解毒这些化合物的酶系统以及限速步骤有一个基本的了解，是使强大的植物系统能够在原位有效地控制和修复爆炸物污染的必要条件。在植物中，大部分TNT留在根部，在那里它抑制生长和发育，减少整个植物生物量。我们最近发现，线粒体和质体靶向酶单脱氢抗坏血酸还原酶6 （MDHAR6）通过一个电子还原TNT，形成与大气氧反应的硝基，产生高活性超氧化物。这种无用的催化循环只需要催化量的TNT就可以在线粒体中不断产生有害的活性氧。我们已经证明MDHAR6的突变体显著增强了TNT的耐受性，我们认为这种反应几乎完全解释了植物对TNT的毒性。这项研究方案的主要目标是严格和定量地确定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毒性的机制，并充分阐明TNT诱导的解毒途径，包括糖基化、谷胱甘肽化和细胞色素P450的氧化活性。由这些酶产生的TNT代谢物的命运将被确定。我们之前已经证明，TNT活性谷胱甘肽转移酶GST-U25可以去除一个硝基，使芳香环更容易被生物降解。我们现在有了GST-U25的详细结构信息，这将使我们能够设计和提高对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植物系统，以清理受污染的场所并防止爆炸物污染水源。

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.