Award details

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

Reference	BB/H006281/1
Principal Investigator / Supervisor	Professor Gregory Challis
Co-Investigators / Co-Supervisors	Dr Sarah Barry
Institution	University of Warwick
Department	Chemistry
Funding type	Research
Value (£)	330,761
Status	Completed
Type	Research Grant
Start date	01/01/2010
End date	31/12/2012
Duration	36 months

Abstract

Thaxtomins are toxins produced by several pathogenic Streptomyces species. They consist of a diketopiperazine core formed by condensation of L-4-nitrotryptophan and L-phenylalanine. Thaxtomin A is produced by Streptomyces scabies and causes common scab in potatoes and other root vegetables by inhibiting cellulose biosynthesis. The thaxtomins are noteworthy for the unusual nitro group on the tryptophan ring and the biosynthetic origin of this group has been the focus of much recent interest in this family of natural products. Commonly nitro groups are introduced biosynthetically by the sequential oxidation of an amino group. In this case however genetic studies suggest that tryptophan is nitrated directly by the cytochrome P450 (CYP450) TxtE and the nitric oxide synthase TxtD provides NO, which is a substrate for the CYP450 catalysed reaction. CYP450s are ubiquitous heme-dependent enzymes which activate molecular oxygen to catalyse a variety of transformations. NO is usually an inhibitor of CYP450s as it binds to ferrous and ferric heme. It is intriguing that a CYP450 might use NO, a common inhibitor, to carry out a transformation and it raises several interesting questions. Our biochemical characterisation of this fascinating member of the CYP450 family has shown that it is responsible for regiospecific nitration of L-tryptophan. We propose to further investigate the substrate specificity and mechanism of this enzyme. We have postulated that the transformation requires the reaction of NO and oxygen, mediated by the heme to form peroxynitrite. Comparison of TxtE with other members of the CYP450 family has revealed important differences in highly conserved regions which may cause structural changes and result in the difference in function of this enzyme. This work will lead not only to further understanding of what is at the moment a unique enzyme but also potentially a deeper understanding of the factors which influence CYP450 reactivity in general.

Summary

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.

Impact Summary

The first manuscript on TxtE, the object of study in the proposal, is currently in preparation. As TxtE is the first example of a cytochrome P450 that has evolved to catalyze a specific nitration reaction, we expect the paper to be published in a high profile journal and will therefore work with our press office and scientific publishers in our usual manner to ensure that we maximise the impact of this publication. Part of our proposed research programme is focused on investigating whether we can exploit TxtE and its partner nitric oxide synthase TxtD to make a derivative of the clinically-used antibiotic daptomycin. We have already contacted Dr Richard Baltz at Cubist Pharmaceuticals, who developed daptomycin and market it in the USA, to explore interest in the derivative. Dr Baltz has confirmed in an email that 'Cubist could help evaluate whether it has commercial potential'. Thus if we are successful in producing the daptomycin derivative, we will, through Warwick Ventures, set up a collaboration with Cubist or other potential UK industrial partners to investigate its clinical utility. Another part of our proposed research programme aims to elucidate the substrate tolerance of TxtE. If we can show that TxtE has broad utility as a nitration catalyst, we will explore with Warwick Ventures the possibility of filing a patent and licensing the invention to fine chemicals manufacturing companies. An obvious candidate would be Biosynth (www.biosynth.com), a Swiss company that makes and sells several tryptophan and indole derivatives. The mechanistic insight generated by this project is likely to contribute to increased understanding of cytochrome P450 catalytic mechanisms in general. Cytochrome P450s are important biocatalysts with diverse potential biotechnological applications and our findings are likely to positively-impact future attempts to harness these enzymes for industrial uses. We will therefore make certain that our mechanistic findings are disseminated as widely as possible to ensure all likely beneficiaries are aware of them through conference presentations, high profile publications and press releases. Sarah Barry, the named researcher co-investigator on this project, has already demonstrated strong capability as a scientist and the potential to go on to a career in academia or science-based industry. The project will further develop her existing skills in chemical synthesis and analysis, molecular genetic manipulation and protein overproduction / purification. It will also develop new skills in mechanistic enzymology. The training provided by the project will provide her with a strong platform to progress in her scientific career.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Industrial Biotechnology, Microbiology
Research Priority	Synthetic Biology
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/H006265/1 Elucidating and exploiting cytochrome P450 TxtE-catalysed tryptophan nitration in thaxtomin phytotoxin biosynthesis

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