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Title: Synthesis of cobalamin analogues using enzymatic and chemical modification methods, and subsequent identification of cobalamin localisation in a variety of organisms
Author: Nemoto-Smith, Emi H.
ISNI:       0000 0004 6349 2911
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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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, ΔmetE, 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 ΔmetE 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.
Supervisor: Warren, Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available