Unravelling the remarkable synthesis and mechanisms involved in the biogenesis of heme and heme d1 from siroheme

This project aims to have a major impact on our understanding of the synthesis and evolution of a related family of heme-like molecules, and will result in a comprehensive addition to text book sections dealing with porphyrin metabolism. Hemes belong to a family of essential life pigment involved in many basic respiratory processes. This application aims to decipher a twenty-year mystery by unravelling the pathway leading to their synthesis. Specifically we aim to elucidate how heme and heme d1 are made in the Archaea and a number of eubacteria. Heme was previously thought to be synthesized along a single pathway from a compound called uroporphyrinogen III by a series of enzymes that modify the acidic side chains, oxidize the macrocycle and insert ferrous iron. However, we have recently demonstrated that heme and a structurally related macrocycle, heme d1, can be made by a completely novel pathway that involves the cannibalism of another modified tetrapyrrole, a compound called siroheme, the prosthetic group of sulphite and nitrite reductase. There are many novel features to this project that make it compelling. The transformation of siroheme into heme and heme d1 requires a number of radical SAM enzymes coupled with a decarboxylase, involving some highly unusual steps that are unprecedented in biological chemistry. The aim of this project is to understand in molecular detail the step-by-step synthesis of hemes along this pathway. Although the d1 heme is only found in the cytochrome cd1, the importance of this enzyme is becoming ever more evident as it vital for the environmentally important ANAMMOX process whereby ammonia is oxidised by nitrite under anaerobic conditions and has recently been implicated in playing a key role in the anaerobic oxidation of methane. It is believed that d1 heme has been adopted for respiratory nitrite reduction because it is tuned to permit nitrite reduction only as far as nitric oxide and not to allow the onward reduction to ammonia that is catalysed by siroheme. The discovery that siroheme is also a precursor for d1 synthesis therefore raises many interesting evolutionary and regulatory questions. Overall, the outcome of this research will result in the composition of a molecular overture of events for a new heme synthesis route, explain more clearly the relationship between heme and siroheme and provide a neodarwinistic understanding of complex biosynthetic pathways. It will also help maintain the UK at the cutting edge of biomolecular science research and maintain the position our groups have as world leaders in this field.

Modified tetrapyrroles play essential roles in many different molecular processes. Despite their prevelance, there is still much to learn about the biosynthesis of these macrocycles. In a whole kingdom of life (the Archaea and also sulfate reducing bacteria) we do not even know how heme is made, but we know that it must be made along an alternative heme biosynthetic pathway since the genes from the classic pathway found in eukaryotes, for instance, are missing. In this application we plan an investigation into the relationship between the biosynthesis of siroheme, heme d1 and heme. We have recently shown that siroheme is, in fact, an intermediate in the synthesis of both heme and d1 heme and we have outlined a testable hypothesis to investigate this further. Previous research had identified a gene cluster responsible for heme d1 synthesis, which includes nirDL, E, F, G, H, J. More recently we undertook a bioinformatics comparison of the Archaea and the sulfate reducing bacteria and identified four genes that are likely involved in the alternative heme biosynthesis pathway (ahbA-D). Surprisingly, we found similarity between the genes of d1 and the alternative heme synthesis, indicating either commonality in some of the intermediates or similarity in the mechanisms employed. The research outlined in this application aims to investigate the individual steps involved in the transformation of siroheme into both d1 and heme. The initial step sees the decarboxylation of siroheme into a compound called didecarboxysiroheme (DDSH). Thereafter, DDSH undergoes a range of modifications to its peripheral side chains to give either heme or d1. These steps involve some unusual chemistry, which will be investigated during the course of the project.

The research described in this application will have a major impact on several areas of science, including synthetic, chemical and systems biology. The basic findings of our preliminary research challenge some existing dogmas about how metabolic pathways and enzymes function. Thus we have demonstrated that heme can be made via a completely novel route, which shares some similarity with the synthesis of heme d1. As such the results will be incorporated into future text books. The research also describes a simple but highly efficient method for the isolation of pathway intermediates, thereby allowing the elucidation of metabolic pathways in a fairly rapid timescale. This method will be particularly applicable to pathways involving labile compounds. With an increase of interest especially of secondary metabolites such an approach is likely to prove popular with chemical biologists and medicinal chemists alike. The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. In essence, the project employs the re-design of existing, natural biological systems for useful purposes. The research also has the potential engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will have application in the biomedicine and bioprocessing of pharmaceuticals and nutrient. The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated, but it will also provide greater insight into the biosynthesis of modified tetrapyrroles. As such, the novel enzyme activities that are involved in the d1 assembly pathway may also be relevant to assembling other kinds of cyclic molecules. If this proves to be the case then we will ensure that our findings are widely disseminated, through example short review articles. Furthermore, there is no doubt that d1 heme is vital for the operation of the Anammox process which is key for waste water treatment. 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. The Warren and Ferguson groups are heavily involved in outreach programmes, through interactions with local schools and community groups. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues. 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 chance to contribute towards a basic understanding of bacterial physiology. 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 Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the new Centre for Molecular Processing within the University of Kent, the research will be brought to the attention of many leading industrial companies.