Investigations into unprecedented reactions associated with the biosynthesis of hemes

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Tetrapyrroles and modified tetrapyrroles have essential roles in a large of range of different molecular processes. We have a far from complete understanding of the reactions, some unprecedented, that biology uses to synthesise these molecules. Recently we discovered an alternative biosynthetic route to heme via double decarboxylation of siroheme, to give didecarboxysiroheme (DDSH), followed by two S-adenosylmethionine (SAM)-dependent reactions that lead to heme via Fe-coproporphyrin IX. Even more recently it has been reported that Fe-coproporphyrin IX can be produced, not from siroheme but from an intermediate, coproporphyrinogen III, on the long established heme synthesis pathway. In this case the required replacement of two propionate groups by two vinyl groups, to give heme, is SAM-independent. Either route for forming the vinyl groups poses novel mechanistic challenges and we shall use a range of contemporary methods to elucidate these unprecedented reaction mechanisms, along with simultaneous interrogation of how coproporphyrinogen III is converted to coproporphyrin IX. Our discovery of a pathway to heme via siroheme and DDSH stemmed from showing that this reaction sequence was also on the pathway to the modified tetrapyrrole, d1 heme, of the periplasmic bacterial cytochrome cd1 nitrite reductase that catalyses the reduction of nitrite to nitric oxide. We now intend to elucidate how the cytoplasmic NirJ protein, a radical SAM enzyme, generates two carbonyl groups at the expense of two propionates and how the periplasmic NirN protein complete the synthesis of the d1 cofactor by introducing a double bond into a propionate side chain. NirN is able to remove two hydrogens from a saturated carbon-carbon bond without dehydrogenation specific cofactors and allows the electrons from the oxidation to flow to heme centre irons in NirN. We will apply a multidisciplinary approach ranging from regiospecific labelling studies through to structural biology and EPR.

The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. The research will show how pathways and enzymes can be investigated by a multi-disciplinary approaches to challenging pathways and will also provide greater insight into the biosynthesis of both a modified tetrapyrrole d1 heme, as well as heme itself. The novel enzyme activities that are involved in the d1 assembly pathway and in newly recognised alternative heme biosynthesis pathways are expected also to be relevant to assembling other kinds of cyclic molecules that can be of interest to the pharmaceutical industry. Thus we will ensure that our findings are widely disseminated, through for example timely short review articles. Notably there is no doubt that d1 heme is vital for the operation of the bacterial Anammox process which is key for waste water treatment because it removes dissolved amonium under anaerobic conditions. Thus we will ensure that our findings are disseminated to those working in waste water treatment. Knowledge of d1 assembly is important for that field as if it were inadvertently inhibited then clearly the process would be blocked. On the other hand, blocking this activity would be relevant to understanding how Pseudomonas aeruginosa, which forms anaerobic biofilms, might be controlled during infections. Thus we will also publicise our findings to the medical field by presenting work at meetings dealing with infection. Thus nation's health could benefit in the longer term from this research.
The Warren and Ferguson groups are heavily involved in outreach programmes, through interactions with local schools and community groups and offering placements to 6th formers which are much in demand from local schools.
The skills acquired by those involved in this project include not only a wide range of important biological techniques ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology but also the opportunity to contribute towards a basic understanding of bacterial physiology. The latter is an area in which there is an increasing knowlege gap in the UK which this type of project will contribute to closing. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Offices. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the Isis Office at Oxford and the new Centre for Molecular Processing within the University of Kent, the research will be brought to the attention of many leading industrial companies.