Synthesis of Cobalamin Analogues Using Enzymatic and Chemical Modification Methods, and Subsequent Identification of Cobalamin Localisation in a Variety of Organisms

Cobalamin, also known as vitamin B12, is an essential nutrient for many different organisms including mammals, fish, birds, nematodes, and a variety of bacteria. However, cobalamin is only synthesised by a few bacteria and archaea. Organisms that cannot synthesise cobalamin de novo must obtain it from their diet. In humans, the cobalamin uptake mechanism has been studied in detail, but in many organisms, such as Caenorhabditis elegans, no method of transport has been defined, and their need for cobalamin is recognised by a cobalamin deficiency phenotype. Corrin ring modified fluorescent analogues of cobyric acid and ribose conjugated fluorescent analogues of cobalamin were synthesised in order to follow the uptake and localisation of these corrinoids in a variety of organisms. Both the C5 corrin-ring modified and the ribose conjugated analogues were absorbed by Salmonella enterica, using the B12 uptake system (Btu) and could be converted into active coenzyme forms. The imaging of these fluorescent analogues enabled the identification of the coelomocytes in C. elegans as a possible storage cell for cobalamin. However, the C5 cobyric acid analogue was not recognised which suggests that the C. elegans cobalamin transport mechanism is specific for complete corrinoid molecules. Lepidium sativum, garden cress, was shown to take up both cobalamin analogues from the roots and store it in the vacuoles of the cotyledons in seedlings, even though plants have no cobalamin requirement. In contrast, Arabidopsis thaliana did not transport any of the cobalamin analogues. Cobalamin deficiency has been implicated in impeding disease progression in a number of diseases, such as tuberculosis. The Mycobacterium tuberculosis cobalamin uptake protein, BacA, has only recently been identified, and there is still much to learn about the relationship between M. tuberculosis and cobalamin. Incubations of a cobalamin dependent strain of M. tuberculosis, ?metE, with a selection of cobalamin biosynthesis intermediates showed that cobyric acid is the earliest intermediate to be taken up and converted into the cofactor form. The C5 corrin ring modified cobyric acid fluorescent analogue is also capable of rescuing this ?metE strain, and is taken up faster than the ribose conjugated cobalamin analogue. Overall, the research outlined in this thesis demonstrates that fluorescent corrinoid analogues can be used to follow the journey of cobalamin in a broad range of different organisms and systems

Synthesis of Cobalamin Analogues Using Enzymatic and Chemical Modification Methods, and Subsequent Identification of Cobalamin Localisation in a Variety of Organisms

Cobalamin, also known as vitamin B12, is an essential nutrient for many different organisms including mammals, fish, birds, nematodes, and a variety of bacteria. However, cobalamin is only synthesised by a few bacteria and archaea. Organisms that cannot synthesise cobalamin de novo must obtain it from their diet. In humans, the cobalamin uptake mechanism has been studied in detail, but in many organisms, such as Caenorhabditis elegans, no method of transport has been defined, and their need for cobalamin is recognised by a cobalamin deficiency phenotype. Corrin ring modified fluorescent analogues of cobyric acid and ribose conjugated fluorescent analogues of cobalamin were synthesised in order to follow the uptake and localisation of these corrinoids in a variety of organisms. Both the C5 corrin-ring modified and the ribose conjugated analogues were absorbed by Salmonella enterica, using the B12 uptake system (Btu) and could be converted into active coenzyme forms. The imaging of these fluorescent analogues enabled the identification of the coelomocytes in C. elegans as a possible storage cell for cobalamin. However, the C5 cobyric acid analogue was not recognised which suggests that the C. elegans cobalamin transport mechanism is specific for complete corrinoid molecules. Lepidium sativum, garden cress, was shown to take up both cobalamin analogues from the roots and store it in the vacuoles of the cotyledons in seedlings, even though plants have no cobalamin requirement. In contrast, Arabidopsis thaliana did not transport any of the cobalamin analogues. Cobalamin deficiency has been implicated in impeding disease progression in a number of diseases, such as tuberculosis. The Mycobacterium tuberculosis cobalamin uptake protein, BacA, has only recently been identified, and there is still much to learn about the relationship between M. tuberculosis and cobalamin. Incubations of a cobalamin dependent strain of M. tuberculosis, ?metE, with a selection of cobalamin biosynthesis intermediates showed that cobyric acid is the earliest intermediate to be taken up and converted into the cofactor form. The C5 corrin ring modified cobyric acid fluorescent analogue is also capable of rescuing this ?metE strain, and is taken up faster than the ribose conjugated cobalamin analogue. Overall, the research outlined in this thesis demonstrates that fluorescent corrinoid analogues can be used to follow the journey of cobalamin in a broad range of different organisms and systems

Construction of Fluorescent Analogs to Follow the Uptake and Distribution of Cobalamin (Vitamin B 12 ) in Bacteria, Worms, and Plants

Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogues with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogues were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogues, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B12

Modification of bacterial microcompartments with target biomolecules via post-translational SpyTagging

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational ‘click’ methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12‐Cofactors for Catalysis

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt-ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt-ions. Consequently, the corrin ligand coordinates cobalt-ions in de-symmetrized ‘entatic’ states, thereby promoting the activation of B12-cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogs of B12

Modification of bacterial microcompartments with target biomolecules via post-translational SpyTagging

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational ‘click’ methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents