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.

Beneficiaries will include biotechnologists wanting to exploit metabolic pathways for the production of existing and new molecules and systems biologists seeking to model metabolic processes. Enzyme concentration becomes very important when the substrate for a reaction is the enzyme-product complex of the previous step, rather than the free product. An improved understanding the transfer of the product of one step, to the next enzyme in the pathway could therefore have important consequences for the industrial exploitation of biosynthetic pathways. We anticipate the basic science being in place at the end of this 48 month project. Biotransformations are important for providing the vitamins, drugs, and other molecules needed to support healthy lifestyle and healthy ageing. Additionally, biosynthetic pathways have important impact on UK food security and on reducing our dependence on oil-based molecules. Synthetic biology approaches to manipulate enzyme concentrations may be important in realising the industrial potential of this research, as may be engineering the recognition events between enzymes and the triggers for product release. Higher product yields may be one benefit of this research bringing clear advantages to our industrial competitiveness and importantly providing the vitamins, drugs, and possibly enhanced food yields needed for the health and well-being of UK citizens in the twenty-first century.