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 -精氨酸的连续氧化生成一氧化氮（NO）。一氧化氮合酶编码基因已经在大量的需氧细菌中被发现，然而，只有少数已经被生物化学表征。一氧化氮合酶（TxtD）已被证明可以产生NO，作为植物病原体疥疮链霉菌产生的天然产物植物毒素萨克托霉素a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，以产生l -4-硝基色氨酸，这是thaxtomin a生物合成的关键组成部分。最近，来自不同链霉菌菌株的其他细胞色素p450也被证明具有硝酸盐色氨酸的作用。这些酶产生不同的区域异构体，5-硝基色氨酸。NOS与细胞色素P450的结合似乎是天然产物生物合成中一种重要的硝化方法，但对其了解甚少。到目前为止，对酶促硝化的研究主要集中在硝化细胞色素p450上，如TxtE，并使用合成的NO供体在硝化反应中提供NO。为了进一步了解这种生物化学，我们有兴趣在体外研究NOS与细胞色素P450的催化结合。因此，我们对生物合成一氧化氮合成酶TxtD进行了过表达纯化，并首次利用TxtD向细胞色素P450提供NO，促进了硝基色氨酸的合成。我们激动人心的初步研究为我们提供了一个极好的机会来进一步研究这种迷人的和生物学上重要的转化，并了解这些酶是如何共同起作用的。这一过程很有趣，因为区域选择性硝化是一个具有合成挑战性的反应。硝基是合成复杂分子（如药物）的关键官能团。然而，合成的硝化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，我们的目标是在初步成功的基础上，充分表征NOS/CYP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，但涉及诸如抗菌素耐药性，植物-微生物相互作用和生物降解等重要过程。进一步了解这些酶将对生物技术、健康和农业产生影响。由于我们对这个系统的了解和我们重要的初步数据，我们将迅速产生高影响力的可发表数据。我们还提出了公众参与活动，以便向公众传播这一基础科学。该提案与BBSRC在“合成生物学”和“工业生物技术的新战略方法”方面的战略重点有关。

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".