Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-␣ subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Å resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded ␤-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.erythropoietin ͉ dioxygenase ͉ hypoxic response ͉ 2-oxoglutarate I n metazoans the ␣͞␤ heterodimeric hypoxia-inducible transcription factor (HIF) (1) regulates the transcription of an array of genes including those coding for glycolytic enzymes, erythropoietin, and VEGF. The levels and transcriptional activity of the HIF-␣, but not the HIF-␤, subunit are regulated by oxygen. Hydroxylation of either Pro-402 or Pro-564 in human HIF-1␣ (2, 3) within the C-terminal oxygen-dependent degradation domain (CODDD) enables its binding to the von Hippel-Lindau protein (pVHL), a targeting element of the E3-ubiquitin ligase; subsequent ubiquitylation leads to proteasomal degradation of HIF-␣ (for reviews, see refs. 4 -7). In humans, this mechanism is augmented by hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain (8); this modification blocks interaction of HIF-1␣ with the CBP͞p300 coactivator, thereby disabling HIFmediated transcription.Hydroxylation of HIF-1␣ is catalyzed by four 2-oxoglutarate (2OG) dioxygenases: three prolyl hydroxlyases (PHD 1, 2, and 3) (also known as HPH 3, 2, and 1 and EGLN 2, 1, and 3; refs. 9-11) and an asparaginyl hydroxylase [factor inhibiting HIF (FIH); refs. 12 and 13]. The available evidence implicates PHD2 as the most important HIF hydroxylase in down-regulating the hypoxic response during normoxia (5,7,14,15).The HIF hydroxylases are Fe(II) and 2OG-dependent dioxygenases (16, 17); their requirement for dioxygen has led to their characterization as cellular oxygen sensors (refs. 9 -11, 18, and 19; Fig. 1a). The first 2OG dioxygenase to be identified was procollagen prolyl-hydroxylase, which like the PHDs catalyzes trans-4-hydroxylation reactions. Pro...

The oxygen-dependent hydroxylation of proline residues in the alpha subunit of hypoxia-inducible transcription factor (HIFalpha) is central to the hypoxic response in animals. Prolyl hydroxylation of HIFalpha increases its binding to the von Hippel-Lindau protein (pVHL), so signaling for degradation via the ubiquitin-proteasome system. The HIF prolyl hydroxylases (PHDs, prolyl hydroxylase domain enzymes) are related to the collagen prolyl hydroxylases, but form unusually stable complexes with their Fe(II) cofactor and 2-oxoglutarate cosubstrate. We report crystal structures of the catalytic domain of PHD2, the most important of the human PHDs, in complex with the C-terminal oxygen-dependent degradation domain of HIF-1alpha. Together with biochemical analyses, the results reveal that PHD catalysis involves a mobile region that isolates the hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. The results will be of use in the design of PHD inhibitors aimed at treating anemia and ischemic disease.

Four compounds in clinical trials for anaemia treatment are potent inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs), but differ in potency and how they interact with HIF at the PHD active site.

Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-KmO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein–coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.