Investigations into the unprecedented reactions associated with the biosyntheses of hemes

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences


The project aims to have a major impact on our understanding of how heme and heme-like molecules are made within bacteria. In so doing the research will impinge upon several important priority areas of BBSRC, including antimicrobial resistance and strategic approaches to industrial biotechnology. This is because in the course of our previous research we have discovered that heme, a major life pigment, is synthesized by at least three distinct routes. The research in this application highlights strategies that can be used to underpin the development of novel antimicrobials through the exploitation of these biochemical differences. Moreover, the transformations involved in the biosyntheses of these hemes involve some very novel chemistry, which if redesigned could be applied to industrial biotechnology. In this respect the research programme involves cross-disciplinary technologies, with the potential to permit the translation of basic discoveries into new products and processes through the development of new biocatalytic entities and pathways.

Our previous research has demonstrated that heme is not made by a single "classic" pathway, rather it can be made either by an "alternative" or a "transitional" pathway. In particular we wish to look at the role of HemY, a recently identified coproporphyrin synthase, that oxidises coprporphyrinogen through the removal of 6 electrons and 6 protons. We have identified a number of inhibitors of this enzyme through specific searches of chemical libraries, but to enhance their development we need to know more about the action of the enzyme. Similarly, two separate enzymes have been identified that convert Fe-coproporphyrin into heme through the decarboxylation of propionic acid side chains into vinyl groups. Such chemistry is, of course, very significant in biotechnology for the conversion of fatty acids into alkenes and thus redesigning these enzyme for fatty acid substrates would have significant industrial relevance. However, to achieve this we must first understand the molecular processes that drive substrate recognition, specificity and catalytic mechanism. The final part of the research is tailored towards an understanding of d1 heme synthesis, where we have make huge progress in delineating the various steps at the molecular level. Of particular interest is the reaction of NirN, which is able to catalyse the introduction of a double bond into one of the propionate side chains within the confines of the periplasm and without the assistance of any flavin or nicotinamide cofactors. Again such chemistry would be of broad interest to the biotechnology sector, especially as a potentially cheap way of reducing alkanes for high-value and platform chemicals. To achieve this long-term goal again we need to determine the precise nature of the reaction and then redesign the system to allow it to be used in other contexts.

Overall, the research addresses basic biochemical questions concerning the biosynthesis of important metabolites within the cell by painting a molecular portrait of events at the molecular level. In so doing it will provide a neodarwinistic understanding of the evolution ofcomplex 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.

Technical Summary

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.

Planned Impact

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 engages with a number of priority areas, from antimicrobial resistance through to industrial biotechnology. In this respect the project applies the engineering paradigm of systems design to metabolism. The research highlights the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will permit the translation of basic discoveries into new products and processes through the development of new biocatalytic entities and pathways.

The beneficiaries of this research will be researchers in academia and industry who are interested in the exploration of biochemical differences and in the redesign of enzymes for biotechnological purposes. 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 heme and hem-like assembly pathways 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.
Description We are starting to understand the molecular processes that drive substrate recognition, specificity and catalytic mechanism in the various pathways for the biosynthesis of heme and d1 heme. we have developed methodologies to determine the precise nature of the reactions involved and in so doing are answering basic biochemical questions concerning the biosynthesis of important metabolites within the cell by painting a molecular portrait of events at the molecular level. Our research has helped in the classification of the now three distinct heme pathways that are found in nature and providing insights into how they may have evolved. The work is of significance especially with the realisation that many pathogenic bacteria utilise a different heme pathway to that found in humans, thereby representing an exploitable difference for the targeting of specific inhibitors. We now have a much clearer understanding of how the pigments of life are made and how they are all related in a biosynthetic manner.
Exploitation Route Much of the chemistry associated with the construction of these molecules would be of broad interest to the biotechnology sector, especially as a potentially cheap way of reducing alkanes for high-value and platform chemicals. Similarly, the different pathways for heme open the possibility of the design of selective inhibitors. There is still much to learn about the evolution of these pathways but the fact that different bacteria use different routes for the construction heme indicates that there is an underlying driver for the adoption of such distinct biosyntheses. There is also a commercial interest in the synthesis of non-animal heme, which acts as a major flavour enhancer for vegetarian food to make it more meat-like in taste. We have been using the knowledge gained from this investigation to apply it to systems to allow for the overproduction of heme and heme-like molecules such that recombinant forms of haemoglobin and leghaemoglobin can be made in large quantities.
Sectors Chemicals,Energy,Healthcare,Manufacturing, including Industrial Biotechology

Description We are working with industrial collaborators who are interested in the feasibility of designing heme biosynthetic inhibitors. Similarly, we are working with others on ways to enhance tetrapyrrole metabolism for key and important enzyme cofactor production. Specifically, our knowledge of pathway engineering is being used by companies interested in the improved production of related molecules such as vitamin B12, which harnesses the early stages of the heme pathway. By syphoning off this early part of the heme pathway in E. coli we have been able to significantly improve the recombinant production of vitamin B12. There is also strong interest in the production of hemoproteins not only for the biotechnology area in the form of P540-type enzymes, but also in the food industry where the addition of heme-containing proteins is being developed to add flavour to vegetarian foods to give them more meat-like flavour. Hence, we have been advising companies on the best way to produce recombinant myoglobin or leghemoglobin.
Sector Chemicals,Manufacturing, including Industrial Biotechology
Impact Types Economic

Description Collaboration with Prof Ligia Sarajevo 
Organisation New University of Lisbon
Department António Xavier Institute of Chemical and Biological Technology
Country Portugal 
Sector Academic/University 
PI Contribution Prof Sarajevo has allowed to explore the heme pathways in a number of pathogenic bacteria that we would otherwise not have studied.
Collaborator Contribution With Prof Sarajevo we have shown that the heme pathway in S. aureus progresses via Fe-coproporphyrin, which is distinct to the other known pathways.
Impact We recently reported on Fe-regulation in S. aureus with a paper in Molecular Microbiology.
Start Year 2014
Description Scientific collaboration with Prof Gunhild Layer 
Organisation University of Leipzig
Department Institute of Biophysical Chemistry
Country Germany 
Sector Academic/University 
PI Contribution By using the technology developed in this application, and through collaboration with Prof Layer, we were able to elucidate the biosynthesis of a related cofactor called coenzyme F430
Collaborator Contribution Prof Layer was able to provide methodology to assist in the formation of active reductase enzymes by reconstitution of Fe-S centres. In so doing we were able to complete our understanding of how F430 is made in methanogenic bacteria.
Impact The research ultimately resulted in a paper in Nature on the biosynthesis of F430
Start Year 2013