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Title: Structural and mechanistic studies on prolyl hydroxylases
Author: Chowdhury, Rasheduzzaman
ISNI:       0000 0004 2735 7093
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2008
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Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.
Supervisor: Schofield, Christopher J. Sponsor: Commonwealth Scholarship Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Chemical biology ; Molecular biophysics (biochemistry) ; Cardiovascular disease ; Protein chemistry ; Chemical crystallography ; Protein folding ; hypoxia inducible factor ; prolyl hydroxylase ; 2-oxoglutarate ; double-stranded-beta helix ; transcription ; nitric oxide ; crystallography