Mutations in isocitrate dehydrogenases (IDHs) have a gainof-function effect leading to R(À)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R-and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC 50 ) values for the R-form of 2HG varied from approximately 25 lM for the histone N e -lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

The biosynthesis of penicillin and cephalosporin antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (IPNS), a non-haem iron-dependent oxidase, catalyses the reaction of a tripeptide, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), and dioxygen to form isopenicillin N and two water molecules. Mechanistic studies suggest the reaction is initiated by ligation of the substrate thiolate to the iron centre, and proceeds through an enzyme-bound monocyclic intermediate. Here we report the crystal structure of IPNS complexed to ferrous iron and ACV, determined to 1.3 A resolution. Based on the structure, we propose a mechanism for penicillin formation that involves ligation of ACV to the iron centre, creating a vacant iron coordination site into which dioxygen can bind. Subsequently, iron-dioxygen and iron-oxo species remove the requisite hydrogens from ACV without the direct assistance of protein residues. The crystal structure of the complex with the dioxygen analogue, NO and ACV bound to the active-site iron supports this hypothesis.

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Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

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...