Does enzyme coupling explain the enigma of cobalt reduction during vitamin B12 biosynthesis?

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Vitamin B12 is the antipernicious anaemia factor that is associated with a number of complex rearrangement and methylation reactions. Its importance in the biosphere can be recognised by our recent finding that more than 50% of all algae species require this vitamin to survive, and acquire the vitamin through a symbiotic relationship with bacteria. The reaction centre of vitamin B12 is the cobalt-carbon bond, which is formed between the centrally chelated cobalt ion of the corrin ring and either an adenosyl group or a methyl group. There is thus some considerable interest in how this bond is made during the biosynthesis of vitamin B12. In this application we wish to explore how this cobalt-carbon bond is formed in the biosynthesis of vitamin B12 (cobalamin). Cobalamin is also one of the most complex 'small molecules' made in biological systems, a complexity that is reflected in the fact that about thirty enzymes are required for its complete de novo synthesis. The unique covalent metal-carbon bond is formed by reacting a cobalt (I) corrin intermediate with ATP. The aim of this grant application is to understand how this cobalt (I) form is made and how it is then presented to the enzyme that transfers the adenosyl group to generate adenosylcobalamin. We have cloned a specific corrin reductase gene and overproduced the encoded enzyme that is responsible for the reduction of the cobalt (II) corrin species to the cobalt (I) form, and have called it CobR. We are the first group in the world to have done this. Moreover, as a result of our preliminary work, we have also crystallised the enzyme and solved its structure. We now wish to investigate how the enzyme works and address how CobR is able to mediate a reaction that appears to be energetically unfavourable. To address this problem, we first plan to investigate the enzymology of the enzyme. We know that it is a flavoprotein and that it should mediate a one electron reduction. Thus it should be possible to detect the enzyme-bound one electron form (a flavosemiquinone) spectroscopically and we have outlined a number of experiments to help us elucidate the catalytic cycle of the enzyme. We also wish to understand how the substrate binds to the enzyme, and to find out if the enzyme mediates it catalytic power by modulating the way in which the substrate binds. We have outlined a number of spectroscopic techniques that will allow us to answer this directly. Finally, our preliminary evidence suggests that CobR interacts with the next enzyme in the pathway, the adenosyltransferase or CobA. We wish to obtain more information on this interaction and have outlined a range of techniques that will allow us to investigate in molecular detail how the two proteins may associate.