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Elucidating and exploiting cytochrome P450 TxtE-catalysed tryptophan nitration in thaxtomin phytotoxin biosynthesis

阐明和利用 thaxtomin 植物毒素生物合成中细胞色素 P450 TxtE 催化的色氨酸硝化

基本信息

批准号：
BB/H006265/1
负责人：
Andrew Munro
金额：
$ 2.23万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2010
资助国家：
英国
起止时间：
2010 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FH006265%2F1
关键词：
Elucidating exploiting cytochrome P450 TxtE

项目摘要

Thaxtomin A is a toxin produced by the bacterium Streptomyces scabies, a plant pathogen which causes common scab in potatoes and other root vegetables. Recent interest in the thaxtomins has focused on the presence of the unusual nitroindole group which is crucial for the toxicity of thaxtomin A. Nitro groups in natural products are rare and where they are present they usually result from the oxidation of an amino group. In the case of thaxtomin however genetic studies by Loria and coworkers have shown that the nitro group is derived from nitric oxide (NO). These studies have suggested that a nitric oxide synthase enzyme produces NO and that another enzyme, a cytochrome P450, is involved in the nitration reaction. In organic chemistry the direct nitration of an aromatic compound requires harsh conditions and is difficult to control leading to mixtures of products. Thus, the discovery of an enzyme which appears to carry out this reaction in a very selective manner is of major interest. Cytochrome P450s (CYP450) are ubiquitous heme-dependent enzymes. They have been found in virtually every mammalian tissue and organ as well as in plants, bacteria, and yeasts. The CYP450 superfamily catalyses a vast array of chemical reactions using molecular oxygen which results in the incorporation of one oxygen atom into the substrate. In bacteria these enzymes are primarily involved in the biosynthesis of natural products where as in mammalian systems CYP450s play a vital role in the metabolism of drugs and toxins in the body. Given their importance in both these areas it is no surprise that CYP450s are still intensively studied more than 40 years after their discovery. CYP450s in mammalian systems are inhibited by NO as it binds to the iron in the heme cofactor thus preventing the enzyme from carrying out catalysis. Since the discovery of nitric oxide synthases in humans, the function of NO in cells has become a major topic of research. The interaction of NO with CYP450s is of increasing importance in this context. Heme dependent nitric oxide synthases have only recently been discovered in bacteria and the function of the resulting NO in the bacterial cell is poorly understood. In the thaxtomin pathway NO is used as a biosynthetic reagent. It is intriguing that a CYP450 uses NO, a common inhibitor, to carry out a chemical reaction and this raises several interesting questions regarding the substrate tolerance, biotechnological utility and mechanism of action of this unusual member of the CYP450 family. Our biochemical studies have revealed that this CYP450 has the ability to directly nitrate an aromatic substrate. This reaction is, to the best of our knowledge, unprecedented for this family of enzymes and significant further investigation is required to determine the scope of this reaction, the mechanism of action of the enzyme and what features determine this radically different reactivity. This work has the potential to reveal further information about the CYP450 family as it may identify additional key structural elements in this enzyme family. Also determining what allows this CYP450 to use NO as a reagent in a chemical reaction while others are inhibited by it will add significantly to the discussion on the effects of NO. Understanding the mechanism by which CYP450s work is of central importance for the future development of drugs (because CYP450s of human metabolism chemically modify drug molecules in the body) and may allow them to be harnessed for furture biotechnology applications. It may also facilitate the development of inhibitors of CYP450s involved in producing toxins in pathogenic bacteria.

Thaxtomin A是由细菌疥疮链霉菌（Streptomyces scabies）产生的毒素，疥疮链霉菌是一种植物病原体，其导致马铃薯和其他根类蔬菜中的常见疮痂。最近对thaxtomin的兴趣集中在不寻常的硝基吲哚基团的存在上，这对thaxtomin A的毒性至关重要。天然产物中的硝基基团很少见，它们通常是由氨基氧化产生的。然而，在thaxtomin的情况下，Loria及其同事的遗传研究表明，硝基来自一氧化氮（NO）。这些研究表明，一氧化氮合酶产生NO，另一种酶，细胞色素P450，参与硝化反应。在有机化学中，芳香族化合物的直接硝化需要苛刻的条件并且难以控制，导致产物的混合物。因此，发现一种似乎以非常有选择性的方式进行该反应的酶具有重大意义。细胞色素P450（CYP 450）是普遍存在的血红素依赖性酶。它们几乎存在于每一种哺乳动物的组织和器官以及植物、细菌和酵母中。CYP450超家族使用分子氧催化大量化学反应，导致一个氧原子掺入底物中。在细菌中，这些酶主要参与天然产物的生物合成，而在哺乳动物系统中，CYP 450在体内药物和毒素的代谢中起着至关重要的作用。鉴于它们在这两个领域的重要性，CYP450在发现40多年后仍被深入研究也就不足为奇了。哺乳动物系统中的CYP 450被NO抑制，因为它与血红素辅因子中的铁结合，从而阻止酶进行催化。自从一氧化氮合酶在人体中被发现以来，NO在细胞中的功能已成为一个主要的研究课题。NO与CYP 450的相互作用在这方面越来越重要。血红素依赖性一氧化氮合酶最近才在细菌中被发现，并且所产生的NO在细菌细胞中的功能知之甚少。在thaxtomin途径中，NO用作生物合成试剂。有趣的是，CYP450使用NO，一种常见的抑制剂，进行化学反应，这提出了几个有趣的问题，关于底物耐受性，生物技术效用和CYP450家族的这个不寻常的成员的作用机制。我们的生物化学研究表明，这种CYP 450具有直接硝酸化芳香底物的能力。据我们所知，该反应对于该酶家族来说是前所未有的，需要进一步的研究来确定该反应的范围、酶的作用机制以及什么特征决定了这种根本不同的反应性。这项工作有可能揭示有关CYP 450家族的更多信息，因为它可能会识别该酶家族中的其他关键结构元件。此外，确定是什么允许该CYP 450在化学反应中使用NO作为试剂，而其他试剂被其抑制，将显著增加关于NO影响的讨论。了解CYP 450的工作机制对于未来药物开发至关重要（因为人体代谢的CYP 450在体内化学修饰药物分子），并可能使它们被用于未来的生物技术应用。它还可能促进参与致病菌毒素产生的CYP 450抑制剂的开发。