The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption ...assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.
AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses ...hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
The oxygen-dependent hydroxylation of proline residues in the α subunit of hypoxia-inducible transcription factor (HIFα) is central to the hypoxic response in animals. Prolyl hydroxylation of HIFα ...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-1α. 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.
Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with ...Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.
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-α 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(ll) and an inhibitor to 1.7 Å resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded β-helix core fold common to the Fe(ll) 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.
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl ...hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35Å) and of Notch peptides bound to FIH (2.4–2.6Å) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.
Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate ...(2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-α. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-α abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-α. Since FIH contains certain motifs present in iron- and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-α fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.
A β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Malassezia globosa has been cloned, characterized, and studied for its inhibition with sulfonamides. This enzyme, designated MG-CA, ...has significant catalytic activity in the CO2 hydration reaction and was inhibited by sulfonamides, sulfamates, and sulfamides with K I in the nanomolar to micromolar range. Several sulfonamides have also been investigated for the inhibition of growth of M. globosa, M. dermatis, M. pachydermatic, and M. furfur in cultures, whereas a mouse model of dandruff showed that treatment with sulfonamides led to fragmented fungal hyphae, as for the treatment with ketoconazole, a clinically used antifungal agent. These data prompt us to propose MG-CA as a new antidandruff drug target.
Studies on hypoxia-sensitive pathways have revealed a series of Fe(ll)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of ...these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IκB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IκBα were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARDcontaining proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.
In humans both the levels and activity of the α-subunit of the hypoxia-inducible transcription factor (HIF-α) are regulated by its post-translation hydroxylation as catalyzed by iron- and ...2-oxoglutarate (2OG)-dependent prolyl and asparaginyl hydroxylases (PHD1-3 and factor-inhibiting HIF (FIH), respectively). One consequence of hypoxia is the accumulation of tricarboxylic acid cycle intermediates (TCAIs). In vitro assays were used to assess non-2OG TCAIs as inhibitors of purified PHD2 and FIH. Under the assay conditions, no significant FIH inhibition was observed by the TCAIs or pyruvate, but fumarate, succinate, and isocitrate inhibited PHD2. Mass spectrometric analyses under nondenaturing conditions were used to investigate the binding of TCAIs to PHD2 and supported the solution studies. X-ray crystal structures of FIH in complex with Fe(II) and fumarate or succinate revealed similar binding modes for each in the 2OG co-substrate binding site. The in vitro results suggest that the cellular inhibition of PHD2, but probably not FIH, by fumarate and succinate may play a role in the Warburg effect providing that appropriate relative concentrations of the components are achieved under physiological conditions.
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