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.
Signalling hypoxia by HIF hydroxylases Schofield, Christopher J.; Ratcliffe, Peter J.
Biochemical and biophysical research communications,
12/2005, Letnik:
338, Številka:
1
Journal Article
Recenzirano
Analysis of oxygen sensitive pathways that regulate the hypoxia inducible factor (HIF) transcriptional system has revealed a novel role for oxygenases in signalling hypoxia. The enzymes, which ...catalyse hydroxylation of specific prolyl and asparaginyl residues in the regulatory HIF-α subunits, belong to the superfamily of non-haem Fe(II)-dependent oxygenases that use the citric acid cycle intermediate 2-oxoglutarate (2OG) as a co-substrate. We review biochemical and physiological data that demonstrate a central role for these oxygenases in integrating multiple signals that coordinate cellular responses to hypoxia.
β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains ...carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant
We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration IC
). MIC assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing
, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against
K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-β-lactam combinations.
Structural studies on human 2-oxoglutarate dependent oxygenases McDonough, Michael A; Loenarz, Christoph; Chowdhury, Rasheduzzaman ...
Current opinion in structural biology,
December 2010, 2010-Dec, 2010-12-00, 20101201, Letnik:
20, Številka:
6
Journal Article
Recenzirano
2-Oxoglutarate and ferrous iron-dependent oxygenases have emerged as an important family of human enzymes that catalyse hydroxylations and related demethylation reactions. Their substrates in humans ...include proteins, nucleic acids, lipids and small molecules. They play roles in collagen biosynthesis, hypoxic sensing, regulation of gene expression and lipid biosynthesis/metabolism. Structural analyses, principally employing crystallography, have revealed that all of these oxygenases possess a double-stranded β-helix core fold that supports a highly conserved triad of iron binding residues and a less well conserved 2-oxoglutarate co-substrate binding site. The 2-oxoglutarate binds to the iron in a bidentate manner via its 1-carboxylate and 2-oxo groups. The primary substrate binding elements are more variable and can involve mobile elements.
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes ...demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
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.
Human isocitrate dehydrogenase (IDH) genes encode for the IDH1, 2 & 3 isoenzymes which catalyse the formation of 2-oxoglutarate from isocitrate and are essential for normal mammalian metabolism. ...Although mutations in these genes in cancer were long thought to lead to a 'loss of function', combined genomic and metabolomic studies led to the discovery that a common IDH 1 mutation, present in low-grade glioma and acute myeloid leukaemia (AML), yields a variant (R132H) with a striking change of function leading to the production of (2R)-hydroxyglutarate (2HG) which consequently accumulates in large quantities both within and outside cells. Elevated 2HG is proposed to promote tumorigenesis, although the precise mechanism by which it does this remains uncertain. Inhibitors of R132H IDH1, and other subsequently identified cancer-linked 2HG producing IDH variants, are approved for clinical use in the treatment of chemotherapy-resistant AML, though resistance enabled by additional substitutions has emerged. In this review, we provide a current overview of cancer linked IDH mutations focussing on their distribution in different cancer types, the effects of substitution mutations on enzyme activity, the mode of action of recently developed inhibitors, and their relationship with emerging resistance-mediating double mutations.
The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates ...including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry- and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.
JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause ...release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344–403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.
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