Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764304
Title: Structural and mechanistic studies of the iron(II)- and 2-oxoglutarate-dependent dioxygenases
Author: Longbotham, Edward
ISNI:       0000 0004 7655 2733
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
The Fe(II)- and 2-oxoglutarate (2-OG)- dependent dioxygenases are a large superfamily of enzymes that catalyse a wide variety of oxidative reactions. They are involved in a range of essential physiological roles, such as DNA repair, antibiotic synthesis and oxygen sensing. Research into these enzymes therefore has implications for the understanding of diseases such as cancer and stroke, which could lead to the design of new drugs, as well as biotechnology applications such as the cheaper and more efficient manufacture of chemicals. In this thesis, two members of this superfamily, a viral collagen prolyl hydroxylase (vCPH) and an asparagine hydroxylase (AsnO) were used as models to investigate the conserved mechanism of these enzymes. Catalytic intermediates were characterised using stopped-flow spectroscopy. In vCPH, the metal to ligand charge transfer (MLCT) complex was characterised and attempts were made to characterise the Fe(IV) intermediate, but they proved unsuccessful. The crystal structure of vCPH was determined for the first time, which showed structural features common to this superfamily of enzymes. A structure was determined with co-substrate 2-OG bound, revealing its mode of binding. In absence of a peptide substrate bound structure, a model was produced of peptide substrate bound in the active site. Catalytic intermediates were also studied in AsnO and a significant isotope effect was found to occur on the decay of the putative Fe(IV) intermediate. An active site variant of AsnO was produced which was hoped would change the activity of AsnO from a hydroxylase to a chlorinase. However, this was determined not to be the case. Catalytic intermediates of this AsnO variant were also determined and compared to the WT AsnO. These data have allowed us to contribute to knowledge about the structural biology and the catalytic cycle involved in this important family of enzymes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.764304  DOI: Not available
Share: