Project overview
Vitamin B12, cobalamin, is just one member of a family of over twenty different related molecules that are called cobamides, molecules that are exclusively made by only certain prokaryotes. What differentiates cobalamin (B12) and makes it a vitamin from these other variants is the presence of an unusual base in the lower nucleotide loop of the cobamide called 5,6-dimethylbenzimidazole (DMB) This project is focussed on how this curious base (DMB) is made under anaerobic conditions. Vitamins are essential micronutrients that are required by cells to perform a diverse range of biological functions, from methylation and complex rearrangement reactions through to light sensing. Vitamin B12 is a cobalt-containing compound that is composed of a corrin ring attached to a lower nucleotide loop. The nutrient is unique among the vitamins in that it is made exclusively by only certain bacteria. The synthesis is orchestrated via a highly complex biosynthetic pathway involving around thirty enzyme-mediated steps. The biologically active forms of the vitamin are most commonly adenosylcobalamin and methylcobalamin, which are involved in rearrangement reactions and as a cofactor for methyltransferases respectively. The nutrient actually belongs to a family of around 20 related molecules that all differ in the nature of the lower ligand to the cobalt, where we typically find benzimidazole derivatives, purine derivatives and aromatics such as phenol. This diversity plays an important role in nutrient availability and acquisition in mixed bacterial communities which include the human microbiome. A key question is why eukaryotes have exclusively selected the form of the cobalamin which contains 5,6-dimethylbenzimidazole (DMB) as its lower ligand over the other twenty variants? In this application we wish to address the synthesis of the base, DMB, found in the lower nucleotide loop. The genes responsible for the anaerobic biosynthesis have been identified but the pathway remains poorly characterised. Surprisingly, bioinformatic analysis of the gene cluster has identified two vitamin B12-dependent radical SAM enzymes. B12-dependent rSAM enzymes represent an understudied, catalytically diverse and incredibly important family of proteins. They form one of the largest groups of enzymes within the rSAM superfamily and have been identified in the pathways of many natural products from bacteriochlorophyll to antibiotics and anticancer agents. Moreover, the presence of B12-dependent enzymes in the biosynthesis of DMB suggests that the vitamin is involved in its own synthesis - in other words B12 is required to make B12. In this program of work, a series of experiments are outlined that will provide an opportunity to address this point and in so doing will provide mechanistic insights into how these enzymes are able to mitigate seemingly impossible reactions. The first three experimental sections of the programme deal with the biochemistry and enzymology of the pathway. In the final section we aim to use this gained knowledge, and apply synthetic biology approaches to develop novel variants of the vitamin. The research will employ recently developed synthetic cofactors and will produce lower base analogues of cobalamin which allow for downstream conjugation with fluorescent molecules or reporter groups. This will generate a tool box of biochemical probes which will be used to improve our understanding of the trafficking of cobalamin, how access to key nutrients can regulate bacterial communities and also provide information on the role of the vitamin in disease processes.
