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. 2019 Jul 26;294(30):11637-11652.
doi: 10.1074/jbc.RA119.008693. Epub 2019 May 30.

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6

Md Saiful Islam  1 , Michael A McDonough  1 , Rasheduzzaman Chowdhury  1 , Joseph Gault  1 , Amjad Khan  1 , Elisabete Pires  1 , Christopher J Schofield  2
Affiliations

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6

Md Saiful Islam et al. J Biol Chem. .

Abstract

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.

Keywords: 2-oxoglutarate and iron dependent dioxygenase; C-5 hydroxylysine; JMJD6; JmjC domain-containing protein 6; RNA splicing; X-ray crystallography; dioxygenase; enzyme catalysis; enzyme structure; hydroxylase; hydroxylysine (Hyl); hypoxia; metalloenzyme; substrate specificity.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Schematic of 2OG-catalyzed hydroxylation and demethylation oxidation reactions. A, outline mechanism of a 2OG-dependent oxygenase catalytic cycle; one atom of O2 is incorporated into the substrate (R–H), a process coupled to the oxidative decarboxylation of 2OG to give succinate and CO2. B, reactions catalyzed by JmjC KDMs and JmjC RDMs, C, JMJD6-catalyzed lysyl C-5 hydroxylation.
Figure 2.
Figure 2.
Evidence that isolated JMJD6 is not a histone N-methyl arginyl demethylase. LC-MS spectra of H4R3(me2s)1–30 (A), H3R2(me2s)1–25 (B), and H4R3(me2s)1–18-K-biotin (C) show the unmodified peptides in the presence of all reaction components except enzyme (black spectra). Red spectra show peaks with +16-Da mass shifts observed in the presence of JMJD6Δ363–403. By contrast, blue spectra show peaks with −14- and −28-Da mass shifts for the JmjC KDM JMJD2E/KDM4E-treated peptides suggesting demethylation. Note the lack of evidence for demethylation in the JMJD6-treated substrates. Adduct, apparent non-enzymatic peptide modification.
Figure 3.
Figure 3.
Evidence that JMJD6 catalyzes hydroxylation of lysine residues from RS-rich regions of SR proteins. MALDI-TOF MS spectra reveal modifications on JMJD6Δ363–403-untreated (black lines) and JMJD6Δ363–403-treated (red lines) peptides from (RS-rich) regions of SR proteins LUC7L2 (A), CROP/LUC7L3 (B), U2AF65 (C), RBM39 (D), Acinus S′ (E), SRSF11 (F), and LUC7L1 (G). Experiments were carried out following standard hydroxylation assay procedures (see under “Experimental procedures” for details).
Figure 4.
Figure 4.
Crystallographic studies of the JMJD6Δ344–403·Fe·2OG complex. A, surface and cartoon representations of a crystal structure of the JMJD6Δ344–403 homodimer (PDB code 6GDY). B, JMJD6 core 8 β-strands of the DSBH fold are highlighted red and labeled as standard roman numerals βI–βVIII (yellow circles); with its cofactor iron shown as an orange sphere, and cosubstrate 2OG and active-site residues shown as sticks. C, views of the JMJD6 iron- and 2OG-binding residues. The left panel shows difference electron density mFo − DFc OMIT map (green mesh) contoured to 3σ. Right panel shows ligand interaction (black dashes) distances in angstroms (blue).
Figure 5.
Figure 5.
Comparison of reported crystal structures for JMJD6. Cartoon representations of high resolution crystal structures of anaerobic JMJD6 in complex with iron and 2OG, JMJD6Δ344–403·Fe·2OG (PDB code 6GDY) (A), compared with aerobic JMJD6 in complex with nickel and acetate, JMJD6Δ344–403·Ni·acetate (PDB code 3K2O) (23) (B), an aerobic FAB fragment, JMJD6, and Fe complex, JMJD6FL·Fe (PDB code 3LD8) (43) (C), and low-resolution aerobic FAB fragment, JMJD6, 2OG, and Fe complex, JMJD6FL·Fe·2OG (PDB code 3LDB) (43) (D) are shown. The insets show differences observed in the 2OG/ligand-binding residues in the different crystal structures. D, left panel shows a comparison of the active sites from PDB structures 3LDB (orange) and 6GDY (green). Middle panel, note the absence of electron density for 2OG in 3LDB compared with Fig. 4C, left panel, showing high resolution structure 6GDY. Act = acetate.
Figure 6.
Figure 6.
Comparison of the JMJD6 crystal structure with those of related 2OG oxygenases. Cartoon representations and domain architectures are shown for JMJD6Δ344–403·Fe·2OG (PDB code 6GDY) (A), compared with JmjC hydroxylases, FIH·Fe·2OG·HIF-1αCAD795–822 (PDB code 1H2L) (B) (24), JMJD5·Co·NOG·RpS6129–144 (PDB code 6F4P) (C) (12) and JmjC demethylases, JMJD2A (KDM4A).Ni·2HG·H3K36(me3)30–41 (PDB code 2YBP) (D) (64), and PHF8(KDM7B)·Fe·NOG·H3K4(me3)1–24 (PDB code 3KV4) (E) (47). In each enzyme, the core 8 β-strands of the DSBH are in gray and labeled (white circles), and other β-strands are in red unless part of a different domain (i.e. PHF8 PHD domain in orange). βIV–V insert regions are highlighted with blue helices. The insets show an expanded view of the active sites of related enzymes comparing the orientation of substrates (yellow sticks and surfaces), metal (spheres) and interacting residues (sticks colored by region as in cartoon and topology). The locations of metal-binding residues highlighted in topology diagrams (right panels) are shown with yellow stars.
Figure 7.
Figure 7.
Comparison of electrostatic surface potential of JMJD6 with those of other 2OG oxygenases. Electrostatic surface potentials for the crystal structures of JmjC hydroxylases are as follows: A, JMJD6Δ344–403·Fe·2OG (PDB code 6GDY); B, JMJD5·Co·NOG (PDB code 4GJZ) (65); C, FIH·Fe·2OG (PDB code 1H2N) (24) and JmjC demethylases; D, PHF8(KDM7B)·Fe·2OG (PDB code 3K3O) (66); E, JMJD2A(KDM4A)·Fe·2OG (PDB code 2GP5) (67). Note the phosphates (from the crystallization buffer) bound at complementary basic regions may indicate locations of nucleic acid phosphate backbone-binding sites.
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