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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


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.

Technical Summary

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.

Planned Impact

In the present project we plan to continue our well-established practices for maximising the impact of the research undertaken. Some specific areas of focus for maximising the impact of this project are outlined below. 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.


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Description This grant was held jointly between Manchester and the group of Prof. Greg Challis at Warwick. The sole PDRA (Dr. Sarah Barry) was based in Warwick, and the costs to Manchester were for small salary components for the Manchester PI (Prof. Andrew Munro), and CoI's Prof. Nigel Scrutton and Dr. Derren Heyes.
The project was focused on structure and mechanism of the cytochrome P450 enzyme TxtE from a Streptomyces bacterium, which catalyzes an unusual nitration reaction. The P450s are heme-containing proteins, and most P450s catalyze the activation and splitting of dioxygen (O2) at the P450 heme iron, leading to insertion of an atom of oxygen into a substrate that binds close to the heme in the P450 enzyme. TxtE goes a step further and reacts nitric oxide (NO) with the oxygen bound to the heme iron (a ferric superoxide intermediate) to form a reactive nitrating species that then goes on to nitrate the substrate by catalysis of the 4-nitration of the amino acid L-tryptophan, producing the product L-4-nitrotryptophan, that is itself then used in the formation of the plant toxin thaxtomin A.
The work done at Manchester involved visits from Dr. Barry and studies using fast reaction kinetic methods to examine the kinetics of the TxtE reaction mechanism, as well as the order in which oxygen and nitric oxide bind to the heme iron. It was concluded that NO (produced by the protein product of an adjacent nitric oxide synthase (or NOS) gene) binds to the heme ferric-superoxide species to generate the reactive nitrating form of the enzyme.
In the major part of the work done by the funded PDRA at Warwick, further studies were done on (i) the mechanism of this unusual enzyme and the order by which the gaseous molecules bind the heme iron; (ii) the demonstration that nitration was not exclusive for L-tryptophan - but could also occur with various analogues of this chemical; (iii) the exploitation of this technology to introduce a nitrated tryptophan into a different Streptomyces antibiotic (the calcium dependent antibiotic, CDA) and to produce a diverse structural derivative of this molecule.
The project thus unravelled the chemical mechanism of a novel member of the cytochrome P450 enzyme family that catalyzes nitration chemistry. It then went further, and proved that this enzyme could be used to produce a nitrated form of a precursor for a different antibiotic - showing how knowledge gained could be applied to generate other novel chemical structures.
Exploitation Route The nitrating enzyme TxtE could be mutated to alter its substrate recognition such that new types of substrates can bind and be nitrated, giving greater chemical diversity of products. These nitrated chemicals could then be used in synthesis of other valuable chemicals.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description The PDRA for this project was based in Warwick. A key aspect of the results generated was the demonstration that a nitrating species generated by the target enzyme (the P450 TxtE) could be used in the synthesis of thaxtomin phytotoxin through nitration of the tryptophan substrate. It was further shown that TxtE could be used to nitrate a different antibiotic molecule, thus demonstrating the potential utility of the P450 in diverse nitration reactions. Other group have shown interest in this technology, notably Frances Arnold's group at Caltech. Work is ongoing to identify categorically the nature of a transient nitrating species (prospectively a peroxynitrite intermediate) responsible for TxtE activity on its tryptophan substrate..
First Year Of Impact 2013
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Description Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2014