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

Thaxtomin A是一种由链霉菌scabies产生的毒素，链霉菌是一种植物病原体，能引起土豆和其他根茎类蔬菜的常见结痂。最近对他红菌素的兴趣主要集中在对他红菌素A的毒性起关键作用的不寻常的硝基吲哚基团的存在上。天然产物中的硝基很少，在存在的地方，它们通常是氨基氧化的结果。然而，在他托明的例子中，洛里亚和他的同事的遗传学研究表明，硝基来自一氧化氮(NO)。这些研究表明，一氧化氮合酶产生一氧化氮，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反应需要苛刻的条件，而且很难控制，导致产物混合。因此，发现一种似乎以非常有选择性的方式执行这一反应的酶是非常有意义的。细胞色素P450(CYP450)是一种普遍存在的血红素依赖酶。它们几乎存在于哺乳动物的每一个组织和器官中，也存在于植物、细菌和酵母中。CYP450超家族利用分子氧催化一系列化学反应，从而将一个氧原子结合到底物中。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，细胞色素P450在药物和毒素在体内的新陈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，难怪细胞色素P450在被发现40多年后仍被深入研究。哺乳动物系统中的CYP450被NO抑制，因为它与血红素辅因子中的铁结合，从而阻止了酶的催化作用。自从在人类中发现一氧化氮合酶以来，一氧化氮在细胞中的作用已成为一个主要的研究课题。在这种情况下，一氧化氮与细胞色素P450的相互作用变得越来越重要。血红素依赖的一氧化氮合酶最近才在细菌中被发现，而细菌细胞中产生的一氧化氮的功能还知之甚少。在thaxtomin途径中，NO被用作生物合成剂。耐人寻味的是，CYP450使用一种常见的抑制剂NO来进行化学反应，这引发了关于底物耐受性、生物技术效用和作用机制的几个有趣的问题。我们的生化研究表明，这种细胞色素P450具有直接硝化芳香族底物的能力。据我们所知，这一反应对这一酶家族来说是前所未有的，需要进行重大的进一步研究，以确定这一反应的范围、酶的作用机制以及是什么特征决定了这种完全不同的反应活性。这项工作有可能揭示更多关于CYP450家族的信息，因为它可能确定该酶家族中的其他关键结构元件。此外，确定是什么允许这种细胞色素P450在化学反应中使用NO作为试剂，而其他酶则被它抑制，这将大大增加对NO影响的讨论。了解细胞色素P450的作用机制对于药物的未来发展至关重要(因为人体新陈代谢的细胞色素P450对体内的药物分子进行化学修饰)，并可能使它们被用于未来的生物技术应用。它还可能促进参与在病原菌中产生毒素的CYP450抑制剂的开发。

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.