Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647521
Title: Evolution of PHDs as oxygen sensors : mechanistic and structural studies of the PHD of Trichoplax adhaerens, the simplest animal, and mechanistic studies of a PHD-like enzyme of the protist Monosiga brevicollis
Author: Boleininger, Anna
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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Abstract:
This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The α/β-heterodimer HIF (Hypoxia Inducible Factor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF α-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 Å), one containing a Trichoplax adhaerens HIFα subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C4-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIFα hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.
Supervisor: Schofield, Christopher Sponsor: Biotechnology & Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647521  DOI: Not available
Keywords: Biochemistry ; Genetics (life sciences) ; Microbiology ; Evolution (zoology) ; evolution ; Trichoplax adhaerens ; oxygen Sensing ; Monosiga brevicollis ; crystal Structure
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