Enzymes as traps in the elucidation of complex biochemical pathways

In this application we outline a method that will allow a step-change in our ability to study complex biochemical pathways, provide molecular detail on fascinating enzyme mechanisms and to rewrite the metabolic control of pathways involving labile intermediates. The elucidation of biochemical pathways is a challenging area that is often complicated by low levels of inherently unstable metabolic intermediates. We have developed a method that allows for the isolation of enzyme-bound metabolites, permitting their characterisation and thereby providing an opportunity to gain atomic resolution of a number of fascinating enzyme-mediated transformations. The application is based on the finding that in some biochemical pathways the product of one reaction is passed directly onto the next in a process known as substrate channelling. Key to this is a tight association between an enzyme and its product, which allows for the isolation of highly stable enzyme-product complexes. We will exploit these properties to unravel the mysteries surrounding the biosynthesis of vitamin B12 (cobalamin). By using His-tagged enzymes of the pathway it is now possible to isolate many of the hitherto ephemeral intermediates, trapped and stabilised on the tagged enzymes as tightly bound enzyme-product complexes. Characterisation of these intermediates will allow the complete elucidation of the corrin pathway. Moreover, a combination of enzymology and X-ray crystallography will permit a detailed understanding of the mechanism of the enzymes that mediate the synthesis of the corrin framework, including the ring contraction process that involves the extrusion of an integral carbon atom in a reaction that has no parallel in nature. Our preliminary data is consistent with the B12 pathway operating by direct metabolite channelling. We outline experiments to investigate this further and to determine whether enzyme rather than substrate concentration controls this metabolic process.

This application outlines research that will have a major impact on the study of metabolic pathways, molecular enzymology and reshape our ideas on how certain metabolic processes function within the cell. This is based on our recent observations that in the cobalamin (vitamin B12) pathway many of the enzymes form very tight enzyme-product complexes, which act to stabilise highly labile pathway intermediates. Such stable product-complexes have not generally been reported before, most likely because they go against existing dogma that enzymes bind substrates and release products, but are obvious in the cobalamin pathway because the intermediates are coloured. In essence, we have used a synthetic biology approach to build partial cobalamin pathway sequences where the terminal enzyme is His-tagged. Purification of the tagged enzyme is associated with the presence of a bound chromphore, which is the reaction product. Using this approach we plan to isolate and characterise all the unknown intermediates in the cobalamin pathway to allow its complete elucidation. We will then use the isolated intermediates to study the enzymology of the individual steps, especially the mechanistically interesting process associated with ring contraction - often described as one of nature's most amazing chemical feats. Finally, we will address the molecular basis of how the enzymes bind their products so tightly, how the products are released by the subsequent enzyme in the pathway and the implication this form of direct metabolite transfer has in terms of metabolic control analysis. The outcomes of this research are likely to result in new concepts in metabolic control processes, which will need to be incorporated into system biology approaches.

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 the idea that all pathways can be described by a series of kinetic constants for individual enzymes comes unstuck if the enzymes behave in a cooperative manner, especially if it involves direct protein-protein interaction. In this respect enzyme concentration becomes more important than substrate concentration and basic Michaelis Menten kinetics no longer apply. Such finding will have serious implications for those working at systems levels, or those in industry interested in optimizing biochemical pathways for metabolite production. 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 vitamin B12. Such knowledge could be used to generate vitamin overproducing bacterial strains to allow its competitive commercial production. Vitamin B12 is also a nutrient that is produced by many pathogenic bacteria and it represents a genuine target for bacteriocidal intervention. The results gained from this research will provide a wealth of molecular data to allow the rational design of such compounds. The Warren group is 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.