ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N6-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH566–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.
The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolylhydroxylation in human hypoxia sensing has ancient origins.T he hypoxia-inducible transcription factor (HIF) is a major regulator of the response to limited oxygen availability in humans and other animals (1-3). A hypoxia-sensing component of the HIF system is provided by 2-oxoglutarate (2OG)-dependent and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydroxylation of HIF-α subunits, a posttranslational modification that enhances binding of HIF-α to the von Hippel-Lindau protein (pVHL), so targeting HIF-α for proteasomal degradation. The HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG oxygenases that catalyze prolyl-hydroxylation, which also includes the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydroxylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hydroxylation of Skp1, a ubiquitin ligase subunit (5-9). The HIF-PHD-VHL triad is likely present in all animals, but probably not in other organisms (3). However, structurally informed bioinformatic analyses imply the presence of PHD homologs in bacteria (10, 11), including in Pseudomonas spp, suggesting PHDs may have ancient origins. ResultsPseudomonas spp. Cont...
SummaryPost-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies.
In prokaryotes and eukaryotes, 2-oxoglutarate (2OG) dependent oxygenases catalyze a wide range of oxidation reactions. [1] With oligomeric substrates, that is, proteins and nucleic acids, their identified reactions are limited to hydroxylations on carbon and N/O-methyl group demethylations through hydroxylation. [2] The factor inhibiting hypoxia-inducible factor (FIH) is a 2OG oxygenase contributing to the regulation of the hypoxic response in animals through bhydroxylation of an Asn residue within the C-terminal transcriptional activation domain of hypoxia-inducible factor 1a (HIF-1a). [3] FIH also catalyzes the hydroxylation of conserved Asn residues in ankyrin repeat domain (ARD) proteins (Scheme 1). [4] ARDs comprise about 33 residues and hydroxylation can increase their conformational stability and may modulate protein-protein interactions. [5] Proteomic and bioinformatic analyses reveal that FIH catalyzed Asn hydroxylation is widespread and that FIH accepts a range of substrate sequences. [4a,b, 5c, 6] Recent work has revealed that FIH hydroxylates sidechains of residues other than Asn; FIH catalyzes the hydroxylation of Asp residues in the human cytoskeletal ankyrin family and His residues in the human tankyrase-2 ARD. [5c, 7] We were interested in testing if FIH can catalyze the hydroxylation of other residues and therefore placed all 20 typical amino acids at the "hydroxylation position" in 20mer peptides, based on a consensus ankyrin (CA) substrate (HLEVVKLLLEHGADVNAQDK) [5b] in which the residue at the potential hydroxylation site (given in bold font) was substituted. The peptides were incubated with FIH under standard conditions and matrix-assisted laser desorption ionization (MALDI) MS was used to identify modifications. In addition to the anticipated hydroxylation of the Asn (> 95 %) and Asp (85 %) containing peptides, modifications were observed for the peptides containing His, Ser, Trp, Leu, and Ile residues (Figure 1, Figure S1, and Table ST1 in the Supporting Information). Shifts of + 16 Da were observed for the Leu (CA_L, about 65 % conversion) and Ile (CA_I, about 25 % conversion) peptides. In contrast, the Ser peptide (CA_S) underwent mass shifts of À2 Da (about 20 %) and À20 Da (about 30 %). Interestingly for the His containing consensus AR peptide (CA_H), we observed not only a + 16 Da shift, corresponding to hydroxylation, [7] but also lower extents of + 14 Da and À2 Da shifts. We observed, to a small extent, a À2 Da mass shift for the Trp peptide. To investigate the relative efficiencies of the oxidation, the CA peptides were assayed simultaneously and then analyzed by LC-MS, for a + 16 Da peak. The following order of efficiency was observed; CA_N @ CA_H % CA_S % CA_D @ CA_I % CA_L ( Figure S2).We investigated modification to the Leu containing CA_L peptide by MS and NMR spectroscopy. MS fragmentation analyses of the product assigned hydroxylation to the Leu (HLEVVKLLLEHGADVLAQDK, Figure S3). 1 H-13 C HSQC NMR analyses of a purified mixture of modified/ unmodified peptides reveals...
Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.
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