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

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.

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.