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

Lead Research Organisation: University of Kent


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.

Technical Summary

The aim of this application is to gain a molecular understanding of the action of the of the co(II)rrinoid reductase CobR of vitamin B12 biosynthesis. This is a key enzyme in the generation of the cobalt-carbon bond of adenosylcobalamin. Initially, the co(II)rrin reductase (CobR), a 18 kDa flavoprotein, generates a co(I)rrin species, which is subsequently adenosylated by the next enzyme in the pathway (CobA). Our research group is the first in the world to have identified a specific gene that encodes CobR. Moreover, we have overproduced the protein, crystallised it and solved its structure. We have done some further preliminary work to show that CobR is amenable to NMR and EPR spectroscopy, since we aim to use these techniques to investigate how the protein binds it substrates and mediates catalysis. The enzyme catalyses an apparently thermodynamically unfavourable reaction, and the overriding aim of the grant is to understand how it accomplishes this task. Initially, we aim to characterise the kinetics of the reaction, using both steady state and fast reaction kinetics, in an attempt to identify reaction intermediates. The spectroscopy approach will allow us to determine whether the corrin substrate or enzyme (or both) undergo conformational change, which may lead to an alteration in the redox potential of the reactants. Our preliminary data also suggests that CobR interacts with the next enzyme in the pathway, the adenosyltransferase (CobA). We will investigate the nature of this interaction and determine, for instance, whether the two enzymes associate to allow direct metabolite transfer of an unstable intermediate. The coupling of these enzymes could help explain how such as unstable entity as a co(I)rrin intermediate could exist in a biochemical pathway.
Description The research funded on this grant allowed the exploration of a key stage in the biosynthesis of vitamin B12, the cobalt containing nutrient that is made exclusively by micororganisms. In this case we studied an enzyme called the corrin reductase (CobR), which is closely linked with the subsequent enzyme in the pathway called the adenosyltransferase (CobA). The reduction has to overcome a lalrge redox potential yet the enzyme (CobR) that catalyses the change in the oxidation state of the metal only has limited power, so we thus had to determine how this enzyme is able to mediate this apparently thermodynamically unfavourable reaction and investigate how it communicates with its partner enzyme CobA. From this research we were able to provide:
1. The kinetic parameters of the co(II)rrin to co(I)rrin reductase (CobR)
2. Molecular detail on the reduction process including the role of key residues within the CobR that are involved in substrate binding
3. Evidence of interaction between the reductase (CobR) and the adenosyltransferase (CobA) and provided molecular detail on how the two proteins combine to allow metabolite transfer.
Exploitation Route The research can be integrated into programmes aimed at providing enhanced levels of cobalamin through genetic engineering approaches. Attempts to integrate this whole process, for instance into plants, which are deficient in B12, will give rise to nutritionally enhanced crops. Such approaches can have enormous impact especially as vitamin B12 deficiency is associated with neural tube defects in unborn babies as well as developmental problems in breast-fed infants. Thus these studies can lead to ways to improvements in diet and well-being.
Sectors Education,Manufacturing, including Industrial Biotechology

Description Our results have provided new molecular detail on the process of cobalt reduction, which is a pre-requisite for adenosylation of the biologically active form of adenosylcobalamin. The identification of this enzyme and the elucidation of the role played by the enzyme has helped in the construction of new strains with enhanced cobalamin production. The high resolution structure of the enzyme has also opened the opportunities for enzyme engineering to allow for the reduction of a number of corrin analogues.
First Year Of Impact 2010
Sector Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic