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.
Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of ...histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.
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.
Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family ...of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.
Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the ...clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual “ping-pong” mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.
Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the α-subunit of hypoxia-inducible factor (HIF) signals for its ...degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1–3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two β-strands (β2 and β3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the β2β3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the β2β3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.
How PHDs achieve specificity: trans-4-prolyl hydroxylation of the transcription factor HIF occurs with stereochemical retention. Substrate-analogue studies show how the von Hippel Lindau tumor ...suppressor protein (pVHL) and the oxygen-sensing hydroxylases (PHDs) achieve specificity for hydroxyprolyl/prolyl residues for the C⁴-exo/endo prolyl conformations, respectively.
Oxygen-sensing mechanisms of eukaryotic multicellular organisms coordinate hypoxic cellular responses in a spatiotemporal manner. Although this capacity partly allows animals and plants to acutely ...adapt to oxygen deprivation, its functional and historical roots in hypoxia emphasize a broader evolutionary role. For multicellular life-forms that persist in settings with variable oxygen concentrations, the capacity to perceive and modulate responses in and between cells is pivotal. Animals and higher plants represent the most complex life-forms that ever diversified on Earth, and their oxygen-sensing mechanisms demonstrate convergent evolution from a functional perspective. Exploring oxygen-sensing mechanisms across eukaryotic kingdoms can inform us on biological innovations to harness ever-changing oxygen availability at the dawn of complex life and its utilization for their organismal development.
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•Current evidence for iron-oxo reactive intermediates is reviewed.•In crystallo intermediates detected in a native extradiol dioxygenase reaction.•Carotenoid cleavage dioxygenases ...catalyse strigolactone biosynthesis.•Identification of plant cysteine oxidases involved in the plant hypoxic response.•Applications of enzyme manipulation to plant biology and agriculture are discussed.
Non-heme iron-dependent oxygenases catalyse the incorporation of O2 into a wide range of biological molecules and use diverse strategies to activate their substrates. Recent kinetic studies, including in crystallo, have provided experimental support for some of the intermediates used by different subclasses of this enzyme family. Plant non-heme iron-dependent oxygenases have diverse and important biological roles, including in growth signalling, stress responses and secondary metabolism. Recently identified roles include in strigolactone biosynthesis, O-demethylation in morphine biosynthesis and regulating the stability of hypoxia-responsive transcription factors. We discuss current structural and mechanistic understanding of plant non-heme iron oxygenases, and how their chemical/genetic manipulation could have agricultural benefit, for example, for improved yield, stress tolerance or herbicide development.
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