Enzymes as traps in the elucidation of complex biochemical pathways

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

Abstract

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.

Technical Summary

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.

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 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.
 
Description There is a serious issue, one that has not previously been satisfactorily addressed, about how large metabolic pathways, often involving unstable intermediates, are able to operate efficiently within the cellular milieu. The release of labile compounds into the cell would make the synthesis very inefficient, unless the pathway is able to channel its metabolites from one enzyme to the next. From this grant we have shown that many of the enzymes involved in the biosynthesis of cobalamin (vitamin B12) retain the product of the reaction they have just catalysed very tightly, thereby forming an extremely stable enzyme-product complex. The bound compound is only released when it comes into contact with the next enzyme along the pathway. This hitherto unobserved property of forming a strong enzyme-product complex has been developed to isolate a number of otherwise short-lived intermediates and we have shown how this property can be exploited to gain a detailed description of a biochemical pathway. This was achieved by using the biosynthetic pathway enzymes as traps to isolate tightly bound enzyme-product complexes. The structures of these pathway intermediates was determined by NMR, after enrichment with 13C-labelled precursors. Crystallisation of the enzyme-product complexes, coupled with enzymology, provided further molecular detail on the processes associated with ring contraction.
The following objectives have so far been achieved:
(1) Molecular detail on the mechanism of corrin ring synthesis in vitamin B12 along the aerobic pathway has been provided
(2) The anaerobic pathway has been elucidated
(3) The enzymology of direct metabolite transfer during cobalamin biosynthesis has been investigated.
Such outcomes are important for understanding how flux along pathways can be modulated to improve productivity.
Exploitation Route 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.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The research 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 main beneficiaries of this research are 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 has not only provided essential information about how pathways and enzymes can be investigated, but it also provides 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.
First Year Of Impact 2014
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Development of cobalamin surrogates as probes and carriers through synthetic and chemical biology approaches
Amount £503,910 (GBP)
Funding ID BB/K009249/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2013 
End 12/2017
 
Description Scientific collaboration with Prof Gunhild Layer 
Organisation University of Leipzig
Department Institute of Biophysical Chemistry
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