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

Journal article

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.

Authors: Islam, MS; McDonough, MA; Chowdhury, R; Gault, J; Khan, A

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Society for Biochemistry and Molecular Biology
• Journal: Journal of Biological Chemistry
• Volume: 294
• Issue: 30
• Pages: 11637-11652
• Publication date: 2019-07-26
• DOI: 10.1074/jbc.RA119.008693
• EISSN: 1083-351X
• ISSN: 0021-9258
• Pmid: 31147442
• Language: English
• Keywords: substrate specificity; JmjC domain-containing protein 6; hydroxylysine (Hyl); dioxygenase; C-5 hydroxylysine; hypoxia; enzyme structure; X-ray crystallography; 2-oxoglutarate and iron dependent dioxygenase; enzyme catalysis; hydroxylase; metalloenzyme; RNA splicing; JMJD6