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

Abstract

In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). 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.

Oxygen-dependent regulation of E3(SCF)ubiquitin ligases and a Skp1-associated JmjD6 homolog in development of the social amoeba Dictyostelium

“…Nevertheless, the ability of proteasomal inhibitors to partially rescue the development of phyA – cells ( Fig. 2 ) indicates that PhyA senses O 2 via an effect on E3(SCF)Ub ligase activity, which is consistent with its evident specificity for Skp1 ( 13 , 54 , 55 ). A possible explanation is that proteasomal inhibition raises the level of FBPs, which would have the effect of driving increased interactions with Skp1 toward levels normally achieved via Skp1 glycosylation.…”

Inhibition of the Oxygen-Sensing Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-Inducible Factor: A Potential Hypoxia Response Modulating Strategy

“…Despite their related but distinct roles in HIF-mediated hypoxia sensing and response, the PHDs and FIH belong to different 2OG oxygenase structural subfamilies, providing an apparent example of convergence to related, but different, functions. The PHDs belong to the prolyl hydroxylase (PH) structural family VIII of 2OG oxygenases, which are typically monomeric and which include other prolyl hydroxylases along with small molecule oxidant oxygenases. , By contrast, FIH belongs to the Jumonji C (JmjC) structural family VI of 2OG oxygenases, − which are often dimeric and which include other protein hydroxylases and N -methyl lysine demethylases acting on histone substrates (demethylation proceeds via initial hydroxylation) …”

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells

“…1A and Table S1). 5,6,7 As an example, C. Left panel shows the primary and secondary coordination sphere of a heme iron-dependent O2 sensing protein, H-NOX (PDB: 3EEE). O2 is stabilized in the structure by a hydrogen-bond triad.…”

“…10,11 The sensing capability of PHD2-like nonheme iron-based enzymatic O 2 sensors is often discussed in terms of their Michaelis constant (K M(O2) ), and varies widely from low micromolar to a millimolar (Table S2). 6 Unlike heme-based O 2 sensors, however, the structural and molecular features that govern the sensing capability of non-heme iron-based O 2 sensors are relatively unexplored. 12,13 This gap in knowledge remains despite the prevalence of non-heme iron-based enzymatic O 2 sensors in bacteria, single-celled eukaryotes, and animals.…”

“…12,13 This gap in knowledge remains despite the prevalence of non-heme iron-based enzymatic O 2 sensors in bacteria, single-celled eukaryotes, and animals. 6,14,15 Iron in PHD2 is coordinated to a 2-oxoglutarate (2OG) cofactor, two histidines and an aspartate from the protein (right panel, Fig. 1A).…”