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Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

DOI: 10.1074/jbc.RA120.013998 DOI Help

Authors: Tongri Liu (University of Oxford) , Martine I. Abboud (University of Oxford) , Rasheduzzaman Chowdhury (University of Oxford) , Anthony Tumber (University of Oxford) , Adam P. Hardy (University of Oxford) , Kerstin Lippl (University of Oxford) , Christopher T. Lohans (University of Oxford) , Elisabete Pires (University of Oxford) , James Wickens (University of Oxford) , Michael Mcdonough (University of Oxford) , Christopher M. West (University of Georgia) , Christopher J. Schofield (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: September 2020
Diamond Proposal Number(s): 12346

Abstract: In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIF╬▒). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Journal Keywords: prolyl-hydroxylase; hypoxia inducible factor (HIF); Dictyostelium discoideum; Toxoplasma gondii; S-phase kinase associated protein 1 (Skp1); hypoxia-inducible factor (HIF); Toxoplasma gondii; dioxygenase; hypoxia; protein evolution

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography