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

DOI: 10.1074/jbc.RA119.008693 DOI Help

Authors: Md. Saiful Islam (University of Oxford) , Michael A. Mcdonough (University of Oxford) , Rasheduzzaman Chowdhury (University of Oxford) , Joseph Gault (University of Oxford) , Amjad Khan (University of Oxford) , Elisabete Pires (University of Oxford) , Christopher J. Schofield (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: May 2019
Diamond Proposal Number(s): 12346

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 heat shock protein 70 (HSP70), oestrogen receptor α (ERα) 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.

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

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography