Metallometabolism; functional characterisation of the cobalamin cobalt chaperone and chelatase CobN S T and W

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

Abstract

Vitamins are essential nutrients required by humans to complete their diet, and by definition are not made within their own body. In many cases vitamins and coenzymes contain micronutrients / rare metals that are difficult for cells to accumulate. In the case of vitamin B12, the antipernicious anaemia factor whose deficiency is associated with a wide range of systems including megablastic anaemia, neurological disorders, and developmental problems in unborn babies, the micronutrient is cobalt. In terms of bioavailability, cobalt is a very precious commodity as it is one of the least abundant metals in Nature. Biological systems therefore have to invest in a good transport system to acquire the metal and once it is obtained the metal has to be presented or chaperoned to where it is required. In this application we wish to explore one important stage in the biosynthesis of vitamin B12. Vitamin B12 is made by a factory of enzymes / in fact in takes about thirty different enzymes to make this essential compound. Enzymes are biological catalysts that speed up reactions that go on inside cells / without enzymes many reactions would take over a decade to occur instead of just a few seconds. We wish to study the insertion of the cobalt ion into vitamin B12. This process is referred to as cobalt chelation. We have identified a protein called CobW that is able to bind cobalt and we believe delivers the metal ion to the enzyme responsible for inserting it into the vitamin. The enzyme that inserts the metal ion is called the cobaltochelatase and in bacteria such as Rhodobacter capsulatus the cobaltochelatase is made up of a three component system with subunits called termed CobN, S and T. We plan to study to study how the metal delivery system (CobW) binds the cobalt and how it presents the metal to the chelatase complex. We will investigte the effect the chaperone has on the activity of the chelatase both inside and outside of the cell. We will also take the opportunity to look at how the chelatase works and determine the role of the various components of the enzyme. This application is aimed at increasing our understanding of how biochemical pathways operate, how they are controlled and how they can be engineered to enhance the metabolic ability of the host cell. From medical, industrial and wealth creation strategic standpoints, this research programme closely follows the remit and aspirations of the BBSRC. The research falls under several major themes of the BMS committee including (A) Fundamanetal Studies of Non-Covalent Interactions Between Molecules, (B) Macromolecular Interactions and their Structural Basis and (D) Biological Catalysis and Biomimetic Chemistry. More specially, the application also addresses a number of BMS priority areas including COMBIOSYS (where our research requires the reconstitution in vitro of a multiple component biochemical pathway as well as the molecular level study of bioactive molecules that operate on a complex biological system), MENZ (where we aim to establish the molecular mechanisms involved in the enzymatic process, including the determination of the nature of any intermediates involved in the reaction and how the enzyme is regulated - which by necessity also involves the determination of the kinetics of the enzymatic reaction including the effect of substrates, inhibitors and activators of the enzyme) and PROLIN (where we aim to understand the detailed interactions between macromolecules and ligands). The application links aspects of biology, chemistry and biophysics.

Technical Summary

Our interest lies in the biosynthesis of natural products and particular in the chemistry of complex metabolic pathways. In order to understand the appearance and evolution of biological pathways, one has to understand both the rules for how organic molecules behave and the logic of metabolism. In this application we plan to investigate the mechanism of the large aerobic chelatase complex associated with vitamin B12 biosynthesis and provide a molecular rationale as to why nature has employed such a system in the cobalamin pathway. One focus will be on how the cell is able to manage its micronutrient content. Typically, micronutrients have to be acquired against large concentration gradients and then delivered to their site of action. In this application we wish to follow the fate of cobalt, which is found only in sparingly soluble forms. We have identified an internal molecular chaperone (CobW), which binds the metal and delivers its to a chelatase complex for its insertion into the corrin ring of vitamin B12. The chelatase complex is itself made from three subunits termed CobN, S and T. The complex inserts the divalent metal ion into the corrin ring in an ATP-dependent manner. There has been no satisfactory explanation for why ATP is required in this process. We now wish to explore the process of metal ion delivery to the chelatase complex and we also wish to investigate the mechanism of cobaltochelation. Within this application we have outlined ways to study this chelation process both in vivo and in vitro. Using this approach we will be able to discern the role played by CobW in the delivery of the metal ion to the chelatase. Moreover, by using metals ions as probes, we will be able to acquire valuable information about the metal ion binding sites as well as the regio and spatial orientation of the subunits within the chelatase. In this respect we will be using small inorganic molecules to help address fundamental issues regarding protein form and function.

Publications

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Graham R (2009) Tetrapyrroles

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Hansson MD (2011) Bacterial ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis ferrochelatase. in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry

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Mendel RR (2007) Metal and cofactor insertion. in Natural product reports

 
Description This grant was investigated the mechanism of the large aerobic chelatase complex associated with vitamin B12 biosynthesis and provide a molecular rationale as to why nature has employed such a system in the cobalamin pathway. A particular focus was on how the cell is able to manage its micronutrient content. Typically, micronutrients have to be acquired against large concentration gradients and then delivered to their site of action. In this work we followed the fate of cobalt, which is found only in sparingly soluble forms. We identified an internal molecular chaperone (CobW), which binds the metal and delivers its to a chelatase complex for its insertion into the corrin ring of vitamin B12. The chelatase complex is itself made from three subunits termed CobN, S and T. The complex inserts the divalent metal ion into the corrin ring in an ATP-dependent manner. There has been no satisfactory explanation for why ATP is required in this process. We explored the process of metal ion delivery to the chelatase complex and investigated the mechanism of cobaltochelation. Within this research we outlined how the enzyme was able to orchestrate the chelation process both in vivo and in vitro. From this we idnetified role played by CobW in the delivery of the metal ion to the chelatase. Moreover, by using metals ions as probes, we were able to acquire valuable information about the metal ion binding sites as well as the regio and spatial orientation of the subunits within the chelatase. Major outcomes included:
1. The EM-derived reconstruction of CobST, which show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers.
2. The substrate specificity of the CobNST complex, where CobN is able to bind both cobalt and either hydrogenobyrinic acid (HBA) or HBA-a,c-diamide. CobST complexes from different organisms are found to activate CobN. Evidence of a complex between CobN, S and T has also been observed for the first time.
3. Cobalt chelation is a rate limiting step in the synthesis of cobalamin, which can be enhanced by the presence of CobW, a protein that acts as a metal chaperone delivery system. Structural work has also revealed the mode of substrate binding in the related cobaltochelatases CbiK and CbiX.
We were also able to provide molecular detail on a number of other chelatases, including the cobaltochelatases CbiX and CbiK. By doing this we were able to provide new insights into the control and regulation of cobalt insertion in the anaerobic pathway.
Exploitation Route The research has provided molecular detail on the process of chelation - giving new insights into key processes such as vitamin synthesis and the production of harm, the red pigment associated with haemoglobin. The research has also highlighted the importance of metal procurement within the cell and how this can be enhanced through the provision of specific metal.transporters. The research can be taken forward for the enhanced production of vitamin B12 and for providing a greater insight into the molecular basis of diseases such a erythropoietic protoporphyria.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The research grant helped provide new techniques at the cutting edge of synthetic biology in the design and redesign of biochemical pathways and as such appeals to two broad beneficiaries - basic scientists trying to understand the nature of fundamental processes within the cell, and applied biotechnologists who are looking for novel findings for exploitation. In terms of basic science this research addresses some simple questions; how is metal processed within the cell and how can it be manipulated to allow for the integration of the metal into a metabolic process? The other beneficiaries of the work would include those in the area of applied biotechnology as well as those involved in the development of enhanced productivity. We have outlined a synthetic biology approach that can be used for any biochemical pathway requiring a metal and thus this method may be of interest to those trying to expand pathways for eg secondary metabolites. Our approach has provided new insights into the biosynthesis of cobalamin, and helped determine how nature is able to coordinate the resourcing of minerals for biosynthesis. The ability to produce larger quantities of cobalamin is being used by an SME to help in the production of amino acids such as methionine. Finally, the data is being used by bio-engineers for the optimization of biochemical pathways.
Sector Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic