Show Abstract ▼

Abstract: Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.

Open Access

Show Abstract ▼

Abstract: The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

Open Access

Show Abstract ▼

Abstract: Post-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.

Show Abstract ▼

Abstract: 2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1, 2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5, 6, 7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9, 10, 11, 12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 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 Nε-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.

Show Abstract ▼

Abstract: Select an isoform: Linking of cosubstrate and substrate binding sites enables highly selective inhibiton of isoforms of human histone lysine demethylases. The results should provide a basis for the development of potent and selective JmjC inhibitors, possibly suitable for clinical use.