A corrin conundrum: Is vitamin B12 required for its own biogenesis?

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.

Vitamin B12 (cobalamin) is an essential nutrient which consists of a corrin ring and a lower base that is attached via the nucleotide loop. The lower base, which is a key distinguishing feature among the cobamides, is 5,6-dimethylbenzimidazole (DMB) in the form of cobalamin selected by eukaryotes. The pathway for the anaerobic biosynthesis of DMB has recently been identified and surprisingly requires two B12-dependent rSAM enzymes (BzaD & E). The synthesis proceeds via the formation of 5-OH-benzimidazole (OH-Bza) from 5-aminoimidazole ribotide (AIR), which is then modified by three SAM-dependent enzymes (BzaC, D & E) to produce DMB. We wish to address a "catch 22" question of whether cobalamin is required for its own biogenesis and investigate whether the cell need to have some cobalamin present before it can make cobalamin for itself. In this project we will investigate the enzymology of the biosynthesis of DMB, evaluate the timing of the attachment of the lower base to cobinamide and uncover the role of B12 in the process. We will try to manipulate the pathway to produce novel lower base derivatives of cobalamin with new chemical functionality. The analogues will be evaluated as potential imaging agents, reporters and drug delivery vehicles. Overall, the objectives of this proposal will be achieved by performing a detailed kinetic study of the enzymes coupled with analysis for the products by HPLC-MS and NMR to determine their structure. Analogues of cobalamin (e.g. rhodibalamin) will be used to further probe the mechanism of the B12-dependent enzymes. Cobalamin variants with a derivatised lower base will be produced by employing cofactor analogues of SAM (allyl or propargyl) which will allow for the attachment of fluorophores or reporter groups. The results of this investigation will unravel the biosynthesis of an unusual substituted benzimidazole and provide valuable insight into the structure and function of enzymes from the B12-dependent rSAM superfamily.

In terms of research findings; the primary beneficiaries will be researchers in academia and industry who are interested in synthetic biology and its applications. The project will generate an assortment of modified vitamins which have potential in the biotherapeutic sector. The research will not only deliver essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the biosynthesis of cobalamin. It will demonstrate how cofactor analogues can be used to introduce new functional groups into natural products and explore the extent to which synthetic biology can be used to functionalise existing small molecule frameworks. This will not only generate a toolkit for metabolic modification but will also provide valuable insights into the molecular recognition and importance of cobalamin by different cellular processes and explore the effectiveness of the vitamin as a carrier molecule. The research falls well within the remit of synthetic biology and is therefore addressing a key priority area.

In terms of staff development; the project will support the establishment of the PI's long-term research programme, seeding the development of a new group which will conduct internationally-leading research into the synthetic biology and mechanistic enzymology of B12 dependent processes and train the next generation of students in the field. The research staff working on this project will be trained in a wide range of techniques spanning molecular biology and protein purification through to synthetic chemistry. These are highly transferable skills which are essential in many biological laboratories and companies exploiting biotechnologies.

In terms of public engagement; we will disseminate the results of the project to the scientific community through publications in high impact journals and presentations at international conferences. We will publish our data, where possible, in open access journals to increase their availability. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The University of Kent is a member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Talks and demonstrations will be given during science week and at schools through direct invitation or arranged through the outreach office. This will ensure good dissemination with the general public.