Kinetic parameters of human aspartate/asparagine-?-hydroxylase suggest that it has a possible function in oxygen sensing.
ABSTRACT: Human aspartate/asparagine-?-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.
Project description:The human 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-?-hydroxylase (AspH) is a potential medicinal chemistry target for anticancer therapy. AspH is present on the cell surface of invasive cancer cells and accepts epidermal growth factor-like domain (EGFD) substrates with a noncanonical (i.?e., Cys 1-2, 3-4, 5-6) disulfide pattern. We report a concise synthesis of C-3-substituted derivatives of pyridine-2,4-dicarboxylic acid (2,4-PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed by using a mass spectrometry-based assay with a stable thioether analogue of a natural EGFD AspH substrate. Certain C-3-substituted 2,4-PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine-carboxylate-related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid in the development of AspH inhibitors suitable for in?vivo use.
Project description: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.
Project description:The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-?-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z'-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.
Project description:Human aspartate/asparagine-?-hydroxylase (AspH) is a 2-oxoglutarate (2OG) dependent oxygenase that catalyses the hydroxylation of Asp/Asn-residues of epidermal growth factor-like domains (EGFDs). AspH is reported to be upregulated on the cell surface of invasive cancer cells in a manner distinguishing healthy from cancer cells. We report studies on the effect of small-molecule active pharmaceutical ingredients (APIs) of human cancer therapeutics on the catalytic activity of AspH using a high-throughput mass spectrometry (MS)-based inhibition assay. Human B-cell lymphoma-2 (Bcl-2)-protein inhibitors, including the (R)-enantiomer of the natural product gossypol, were observed to efficiently inhibit AspH, as does the antitumor antibiotic bleomycin A2. The results may help in the design of AspH inhibitors with the potential of increased selectivity compared to the previously identified Fe(II)-chelating or 2OG-competitive inhibitors. With regard to the clinical use of bleomycin A2 and of the Bcl-2 inhibitor venetoclax, the results suggest that possible side-effects mediated through the inhibition of AspH and other 2OG oxygenases should be considered.
Project description:The post-translational hydroxylation of prolyl and lysyl residues, as catalyzed by 2-oxoglutarate (2OG)-dependent oxygenases, was first identified in collagen biosynthesis. 2OG oxygenases also catalyze prolyl and asparaginyl hydroxylation of the hypoxia-inducible factors that play important roles in the adaptive response to hypoxia. Subsequently, they have been shown to catalyze N-demethylation (via hydroxylation) of N(?)-methylated histone lysyl residues, as well as hydroxylation of multiple other residues. Recent work has identified roles for 2OG oxygenases in the modification of translation-associated proteins, which in some cases appears to be conserved from microorganisms through to humans. Here we give an overview of protein hydroxylation catalyzed by 2OG oxygenases, focusing on recent discoveries.
Project description:The ethylene-forming enzyme (EFE), like many other 2-oxoglutarate (2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative decarboxylation of 2OG to succinate and CO2 to generate a highly reactive iron species that hydroxylates a specific alkane C-H bond, in this case targeting l-arginine (Arg) for hydroxylation. However, the prominently observed reactivity of EFE is the transformation of 2OG into ethylene and three molecules of CO2. Crystallographic and biochemical studies have led to several proposed mechanisms for this 2-fold reactivity, but the detailed reaction steps are still obscure. Here, the thermodynamics associated with iron(II), 2OG, and Arg binding to EFE are studied using calorimetry (isothermal titration calorimetry and differential scanning calorimetry) to gain insight into how these binding equilibria organize the active site of EFE, which may have an impact on the O2 activation pathways observed in this system. Calorimetric data show that the addition of iron(II), Arg, and 2OG increases the stability over that of the apoenzyme, and there is distinctive cooperativity between substrate and cofactor binding. The energetics of binding of 2OG to Fe·EFE are consistent with a unique monodentate binding mode, which is different than the prototypical 2OG coordination mode in other 2OG-dependent oxygenases. This difference in the pre-O2 activation equilibria may be important for supporting the alternative ethylene-forming chemistry of EFE.
Project description:2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(?)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.
Project description:Covering: up to 2017 2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of ?-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of ?-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of ?-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in ?-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in ?-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.
Project description:Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.
Project description:The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. This enzyme is reported to simultaneously catalyze the conversion of 2OG into ethylene and three CO2 molecules and the C? hydroxylation of l-arginine (l-Arg) while oxidatively decarboxylating 2OG to form succinate and carbon dioxide. A new plasmid construct for expression in recombinant Escherichia coli cells allowed for the purification of large amounts of EFE with activity greater than that previously recorded. A variety of assays were used to quantify and confirm the identity of the proposed products, including the first experimental demonstration of l-?1-pyrroline-5-carboxylate and guanidine derived from 5-hydroxyarginine. Selected l-Arg derivatives could induce ethylene formation without undergoing hydroxylation, demonstrating that ethylene production and l-Arg hydroxylation activities are not linked. Similarly, EFE utilizes the alternative ?-keto acid 2-oxoadipate as a cosubstrate (forming glutaric acid) during the hydroxylation of l-Arg, with this reaction unlinked from ethylene formation. Kinetic constants were determined for both ethylene formation and l-Arg hydroxylation reactions. Anaerobic UV-visible difference spectra were used to monitor the binding of Fe(II) and substrates to the enzyme. On the basis of our results and what is generally known about EFE and Fe(II)- and 2OG-dependent oxygenases, an updated model for the reaction mechanism is presented.