Understanding the role of nitric oxide synthase in biosynthetic nitration

点击翻译按钮获取中文摘要

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".