Fig. 1.

JMJD5 catalyses stereoselective C-3 hydroxylation of Arg137 of the 40S ribosomal protein S6 (RPS6). a MALDI-MS spectra for RPS6_129–144 showing the +16 m/z shift following treatment with JMJD5 and cofactors. b MS/MS analysis of the modified RPS6_129–144 indicating a +16 shift on y and b ion fragments y₈–y₁₄/b₉–b₁₅, which is not observed for ion fragments y₁–y₇/b₂–b₈, implying hydroxylation at RPS6 R137; observed/predicted masses for the fragment ions are given in Supplementary Fig. 5. c Plot of succinate formation monitored by ¹H-NMR. Succinate formation from a reaction with 10 μM JMJD5, 50 μM RPS6, 200 μM 2OG and 100 μM Fe(II) (black) is compared to that without RPS6 peptide (blue), with ascorbate (red), and with an inactive H321A variant substituting for wild type (yellow). Results are the mean ± s.e.m. (n = 3). d JMJD5-catalysed arginyl hydroxylation. e Extracted ion chromatograms (m/z = 345) from LC-MS analysis of synthetic (3R)- and (3S)-hydroxyarginine (HyR) standards and amino-acid hydrolysates from unmodified/hydroxylated RPS6_129–144. Peaks at 1.93 and 2.02 min correspond to (3R)- and (3S)-hydroxyarginine, respectively. f¹H–¹³C HSQC spectra of hydroxylated RPS6_129–144 (VPRRLGPKR^OHASRIRKL), labelled with aa assignments, supporting R137 C-3 hydroxylation. Red boxes highlight the hydroxyarginine (R^OH). Inset bottom right: ¹H–¹H COSY spectrum showing the correlation between C-2-R^OH and C-3-R^OH protons. ♯ Indicates residual HEPES buffer