Abstract
In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (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 cellular 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, 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.
Highlights
Data for HsPHD2(181-426) catalyzed hydroxylation of hypoxia inducible factor (HIF)-a C- and N-terminal oxygen-dependent degradation domains (CODD and NODD, respectively) and factor inhibiting HIF (FIH) catalyzed hydroxylation of the HIF-a C-terminal transactivation domain (CTAD) are from Tarhonskaya et al [54, 56]
The results reveal, at least under the conditions tested, that the HIFa ODDs were the only substrates hydroxylated by HsPHD2 (Tables 1 and 2), i.e. HsPHD2 did not hydroxylate any of the S-phase kinase associated protein 1 (Skp1) substrates tested, consistent with a recent study reporting that isolated PHDs are highly selective for HIF-a
Unlike HIF-a ODD substrates of the HsPHDs and HIF-a CTAD substrates of FIH, but like the bacterial prolyl hydroxylase, P. putida PHD (PPHD), which acts on elongation factor thermounstable (EF-Tu) and ankyrin repeat domain FIH substrates, the Skp1 substrates of D. discoideum PhyA (DdPhyA) and T. gondii prolyl-4hydroxylase A (TgPhyA) have ordered core-folds
Summary
Purified DdPhyA and TgPhyA were found to catalyze hydroxylation of their WT substrates, i.e. full-length DdSkp1 and TgSkp1 proteins, respectively, in a 2OG-dependent manner as evidenced by a 116 Da mass shift using ESI-MS (Fig. 3). Consistent with prior assignments, the results reveal that DdPhyA and TgPhyA only catalyze hydroxylation of Pro143 in DdSkp1 and Pro154 in TgSkp1, respectively We investigated the regiochemistry of DdSkp1 and TgSkp1 prolyl hydroxylation by NMR spectroscopy using Skp1 fragment peptide substrates (Fig. S5).
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