Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal <em>Trichoplax adhaerens</em>

Abstract

BackgroundIn humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens.MethodsWe report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals.ResultsHigh resolution crystal structures (≤1.3 Å) of TaPHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons.ConclusionConserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.

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

“…S5 ). Consistent with previous reports, the results confirmed that DdPhyA and TgPhyA catalyze C-4 prolyl hydroxylation, as shown for some, but not all, other prolyl hydroxylases ( 33 , 34 , 52 , 53 ).…”

“…Such an understanding has been hindered by a dearth of structures of PHD type oxygenases that accept non HIF-a substrates. Aside from studies on human PHD and TaPHD (16,50) (34), both of which have HIF-a substrates, there are no reports of structures of eukaryotic PHD homologs, nor on the crossreactivity of prolyl hydroxylases with the (potential) substrates reported from different types of eukaryotes. Here we report biochemical and biophysical studies on the PHD-like enzymes from Dictyostelium discoideum (DdPhyA) and Toxoplasma gondii (TgPhyA).…”

“…Experimental and bioinformatic studies on the evolution and extent of the HIF-PHD-pVHL system across biology ( 3 , 29 , 30 , 31 ) have revealed PHD-type enzymes in a range of nonmetazoan-type eukaryotes ( 9 , 32 ) and some bacteria ( 33 ). Studies on a PHD from the simple animal Trichoplax adhaerens (TaPHD), have led to validation of a functional HIF system in this organism ( 9 , 34 ). A PHD-like enzyme from Pseudomonas putida (PPHD) has been shown to catalyze prolyl hydroxylation of the ubiquitous protein elongation factor-thermally unstable (EF-Tu), implying a role for prolyl hydroxylation in regulation of the translation machinery in bacteria ( 33 , 35 ).…”

“…These in part reflect substantial conformational changes that are required to enable catalytically productive binding of the well-structured EF-Tu to PPHD ( 33 ). By contrast, the HIF-α NODD/CODD regions that are PHD substrates are substantially disordered ( 49 ), although portions of them adopt defined secondary structure when bound to the PHDs ( 16 , 34 , 50 ).…”

Evolution of metazoan oxygen-sensing involved a conserved divergence of VHL affinity for HIF1α and HIF2α

“…PHD cloned from T. adhaerens can function to regulate HIFα when expressed in human cells, suggesting remarkable conservation of this pathway during metazoan evolution 7 . Crystallization of T. adhaerens PHD bound to its corresponding HIFα peptide reveals significant structural conservation with human PHD2 bound to human HIFα peptides 8 . However, despite conservation of the core proteins critical for oxygen-sensing throughout the majority of animal evolution, duplication of the genes encoding these proteins is observed.…”