Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638492
Title: The alternative haem biosynthesis pathway : structure, function and properties of sirohaem decarboxylase
Author: Palmer, David James
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2014
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Abstract:
Haem, a cyclic tetrapyrrole, is found in organisms from all three domains of life. Haem is a prosthetic group for many proteins involved in essential biological processes such as respiration and oxygen transport. Synthesis of haem in eukaryotes and most bacteria follows a well defined route with highly conserved intermediates. However, an alternative haem biosynthesis pathway in Archaea and some bacteria was recently elucidated. This newly discovered pathway utilises sirohaem as a metabolic intermediate rather than a prosthetic group. The alternative haem biosynthesis pathway is catalysed by the Ahb enzymes A, B, C and D. Initial decarboxylation of sirohaem occurs at the two acetic acid side-chains attached to carbons C12 and C18 to give didecarboxysirohaem, a process catalysed by AhbA and AhbB. Subsequently, the radical SAM enzyme AhbC converts didecarboxysirohaem to Fe-coproporphyrin III. Finally, AhbD catalyses the conversion of Fe-coproporphyrin III into haem in another SAM dependent reaction. This study focused on the AhbA and AhbB proteins from three sources, Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri. Purifications of individual recombinantly produced proteins revealed that both AhbA and AhbB are highly unstable. However, low concentrations of D. desulfuricans and D. vulgaris AhbA and AhbB proteins were isolated and were discovered to have decarboxylase activity. Simultaneous overproduction of AhbA and AhbB proteins facilitated the co-purification of stable heteromeric AhbA/B complexes from all three organisms. However, despite their sequence similarities, distinctly different properties were observed between the homologues including differences in oliogmeric state and haem/product binding capabilities. The D. desulfuricans enzyme has been crystallised and its structure has been elucidated in both apo- and product-bound forms. Mutagenesis of the D. desulfuricans complex has provided further information about active site residues and a mechanism of action has been proposed. Finally, novel functional chimeric complexes were produced using the Desulfovibrio proteins.
Supervisor: Warren, Martin J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.638492  DOI: Not available
Keywords: QP517 Biochemistry
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