Understanding the role of nitric oxide synthase in biosynthetic nitration

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".

Recently, for the first time, used a nitric oxide synthase (NOS), recombinant TxtD, to provide nitric oxide (NO) to two different NO dependent nitrating cytochrome P450s (CYP), TxtE and 5-NTSlav, to facilitate regiospecific nitration of L-tryptophan.
TxtD and TxtE are involved in the biosynthetic pathway of the natural product thaxtomin A in Streptomyces scabies. Together, they are responsible for producing the key biosynthetic precursor, L-4-nitrotryptophan. 5-NTSlav was recently discovered to nitrate L-Trp at the 5 position. 5-NTSlav is encoded in the biosynthetic gene cluster of an as yet, unknown natural product, in Streptomyces lavendulae.
This combination of NOS and CYP appears to be an important nitration method in natural product biosynthesis, but is still poorly understood. Investigations into enzymatic nitration thus far, have focused on P450s and use a synthetic NO donor to provide NO during the nitration reaction. Our exciting initial studies, which fully reconstitute the in vivo reaction, give us an excellent opportunity to investigate this fascinating transformation and understand how NOS and CYPs function together. The aims of this proposal are 1) to fully characterise the NOS, TxtD; 2) to understand how NOS and CYPs work together to catalyse nitration; 3) to understand how the proteins react to nitration conditions which can result in nitrosylation and radical nitration of protein residues.
Regioselective nitrating enzymes open up the possibility of developing nitrating biocatalysts. Nitro groups are key functional groups in synthetic chemistry. However synthetic methods for nitration remain non-selective. The results from this work will generate insight into natural product biosynthetic pathways and bacterial NOS biochemistry. NOSs have been implicated in processes from antimicrobial resistance to plant-microbe interactions. Further understanding of these enzymes will thus have an impact on biotechnology, health and agriculture.

The impacts of this work extend from immediate academic impact in a range of disciplines to ultimately industrial; societal and economic benefit. This proposal focuses on investigating the role of nitric oxide synthases and nitric oxide in bacteria. Particularly in natural product biosynthesis. Due to its multidisciplinarity, this work will impact and advance many fields of research, including but not limited to biochemistry, microbiology and synthetic biology. Research results will be disseminated through presentations at national and international conferences by the PI and the PDRA and ultimately through publication in high impact peer reviewed journals which will be open access.
The economic benefit and applications resulting from this project will likely be in the medium to long term. The knowledge we gain on bacterial nitric oxide synthase dependent nitration will be of interest to the biotechnology sector. We will foster links with industry through membership of networks such as BBSRC funded Network in biotechnology and bioenergy (NIBB): Natural Products Discovery and Bioengineering Network; BIOCATNET and the EPSRC funded Dial-a-molecule network thus enabling the development of promising aspects of the project. These partnerships will be entered into with the help of the highly experienced team in the King's Commercialisation Institute which facilitates and accelerates innovation and commercialisation of research within King's College London. More broadly, this work will further the knowledge economy through the training of an excellent postdoctoral scientist and the further understanding of enzymes involved in biosynthetic transformations and how they may be used to facilitate synthetic biology. UK economic competitiveness relies significantly on the development of biotechnology including the use of synthetic biology approaches or the synthesis of bulk chemicals and complex high value compounds such as pharmaceuticals. Thus a fundamental understanding of microbial biochemistry is of vital importance.
The PDRA who is employed to work on this project will benefit from a multidisciplinary training. King's College London provides extensive courses in researcher training and development for transferable skills training for researchers. Furthermore the collaborative nature of the chemistry department at KCL means the PDRA will be exposed to a variety of disciplines from around the college from physics to the biomedical sciences. I am active in my support of undergraduates as future researchers and have hosted undergraduates in summer research fellowships funded by for example the Royal Society of Chemistry and the KCL Experience programme. My group have been extensively involved in public engagement and outreach activities. We will carry on these regular activities over the course of this grant and I will encourage the PDRA to participate in outreach. Specifically we have requested funds to organise a workshop featuring aspects of our research at KCL Science Festival which is open to members of the public as part of National Science Week. Secondly we would like to use the world class facilities of the Nikon Imaging Centre at KCL to generate images of Streptomyces bacteria which are the source of the enzymes in this proposal, for use on our website and for posters, talks and public engagement activity.