Understanding the role of nitric oxide synthase in biosynthetic nitration

本提案的目的是获得一氧化氮合酶（NOS）及其产物一氧化氮在生物合成硝化中的作用的分子理解。NOSs催化L-精氨酸氧化生成一氧化氮（NO）。一氧化氮合酶编码基因已被确定在大量的好氧细菌，然而，只有少数已被生化特性。一氧化氮合酶，TxtD，已被证明产生NO作为thaxtomin A，由植物病原体疥疮链霉菌产生的天然产物植物毒素的生物合成前体。在这种生物合成途径中，NO被不寻常的细胞色素P450 TxtE在区域特异性硝化反应中使用，产生L-4-硝基色氨酸，这是毒蜥球蛋白A生物合成中的关键结构单元。最近，来自不同链霉菌菌株的其他细胞色素P450也被证明可以硝酸色氨酸。这些酶产生不同的区域异构体，5-硝基色氨酸。NOS和细胞色素P450的这种组合似乎是天然产物生物合成中重要的硝化方法，但仍然知之甚少。迄今为止，对酶促硝化的研究集中在硝化细胞色素P450例如TxtE上，并在硝化反应期间使用合成的NO供体来提供NO。为了更好地了解这种生物化学，我们有兴趣在体外研究NOS和细胞色素P450的催化结合。因此，我们已经过表达和纯化的生物合成的一氧化氮合酶，TxtD，并为第一次，使用TxtD提供NO细胞色素P450，以促进硝基色氨酸的合成。我们令人兴奋的初步研究为我们提供了一个极好的机会，进一步研究这种迷人的生物学重要转化，并了解这些酶如何共同发挥作用。该方法是令人感兴趣的，因为区域选择性硝化是合成上具有挑战性的反应。硝基基团是合成复杂分子如药物的关键官能团。然而，硝化的合成方法仍然是非选择性的，通常需要苛刻的条件。区域选择性硝化酶的发现为开发硝化生物催化剂创造了令人兴奋的可能性。在这个项目中，我们的目标是建立在我们最初的成功和充分的NOS/反硝化系统。该项目将产生有关天然产物生物合成途径的重要信息，这些信息将在合成生物学领域引起兴趣。我们将深入了解细菌NOS的生物化学，这些生物化学知之甚少，但与抗菌素耐药性，植物-微生物相互作用和生物降解等重要过程有关。对这些酶的进一步了解将对生物技术、健康和农业产生影响。由于我们对该系统的了解和我们重要的初步数据，我们将快速生成具有高影响力的可重复数据。我们还建议开展公众参与活动，以便向公众宣传这一基础科学。该提案与生物安全与资源中心在“合成生物学”和“工业生物技术新战略方针”方面的战略优先事项有关。

The aim of this proposal is to gain a molecular understanding of the role of a nitric oxide synthase (NOS) and its product, nitric oxide, in biosynthetic nitration. NOSs catalyse the sequential oxidation of L-arginine to produce nitric oxide (NO). Nitric oxide synthase encoding genes have been identified in large numbers of aerobic bacteria, however, only a handful have been biochemically characterised. The nitric oxide synthase, TxtD, has been shown to produce NO as a biosynthetic precursor of thaxtomin A, a natural product phytotoxin produced by the plant pathogen Streptomyces scabies. In this biosynthetic pathway, NO is used by the unusual cytochrome P450, TxtE, in a regiospecific nitration reaction to produce L-4-nitrotryptophan, a key building block in thaxtomin A biosynthesis. Recently, other cytochrome P450s, from different Streptomyces strains, have also been shown to nitrate tryptophan. These enzymes produce a different regioisomer, 5-nitrotryptophan. This combination of NOS and cytochrome P450 appears to be an important nitration method in natural product biosynthesis, but one which is still poorly understood. Investigations into enzymatic nitration thus far, have focused on the nitrating cytochrome P450s e.g. TxtE and use a synthetic NO donor to provide NO during the nitration reaction. In order to understand more about this biochemistry, we are interested in investigating the catalytic combination of NOS and cytochrome P450 in vitro. Thus we have overexpressed and purified the biosynthetic nitric oxide synthase, TxtD, and for the first time, used TxtD to provide NO to cytochrome P450 to facilitate nitrotryptophan synthesis. Our exciting initial studies give us an excellent opportunity to further investigate this fascinating and biologically important transformation and understand how these enzymes function together. This process is of interest as regioselective nitration is a synthetically challenging reaction. Nitro groups are key functional groups in the synthesis of complex molecules such as pharmaceuticals. However synthetic methods for nitration remain non-selective, often requiring harsh conditions. The discovery of regioselective nitrating enzymes creates the exciting possibility of developing nitrating biocatalysts. In this project we aim to build on our initial success and fully characterise the NOS/CYP nitrating system. This project will produce important information about natural product biosynthetic pathways which will be of interest in the field of synthetic biology. We will generate insight into the biochemistry of bacterial NOS which are poorly understood but have been implicated in important processes such as antimicrobial resistance, plant-microbe interactions and biodegradation. Further understanding of these enzymes will have an impact on biotechnology, health and agriculture.Due to our knowledge of this system and our significant preliminary data we will quickly generate high impact publishable data. We have also proposed public engagement activities in order to communicate this fundamental science to the public. This proposal is relevant to BBSRC strategic priorities in "synthetic biology" and "new strategic approaches to industrial biotechnology".