Abstract
Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.
Highlights
Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles
Because JMJD5 has been assigned as a KDM11, we investigated both hydroxylase and KDM activities for recombinant JMJD5 using reported substrate(s) and binding proteins—H3K36me[211,28] and p5317, NFATC121, PKM219 and RCCD120
It is clear that some 2OG-oxygenases catalyse post-translational modification (PTM) to ribosomal proteins and tRNA1,2
Summary
The results reveal a JMJD5-dependent shift of +16 Da on a single peptide comprising residues 134–150 of human RCCD1, which was abrogated by alanine substitution of the arginine at position 141 in RCCD1 (Supplementary Fig. 3 and Supplementary Table 1). This observation suggested the possibility of JMJD5catalysed arginyl-hydroxylation, which is notable, because to date there has been no report of an arginyl-hydroxylase in humans, or eukaryotes. Inset bottom right: 1H–1H COSY spectrum showing the correlation between C-2-ROH and C-3-ROH protons. ♯ Indicates residual
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