Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.
Bottom Line: However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues.NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position.The results further expand the scope of FIH-catalysed hydroxylations.
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.Show MeSH
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Mentions: MS analyses assigning hydroxylation at His 238 and His 553 in tankyrase-2. Tankyrase-2 was purified from transiently transfected 293T cells coexpressing FIH. (A) MS/MS spectra of the tryptic peptide IVQLLLQHGADVHAK derived from tankyrase-2 (residues 226–240) in the hydroxylated ([M + 3H]3+ = m/z 553.28) (upper) and nonhydroxylated ([M + H]3+ = m/z 548.63) (lower) state. The hydroxylated species (upper) exhibits a + 16 Da mass shift on the y-ion series appearing at y3 and assigning hydroxylation to His238. (B) MS/MS of the tankyrase-2 tryptic peptide containing His 553 (VSVVEYLLQHGADVHAK) in hydroxylated ([M + 3H]3+ = m/z 627.64) (upper) and unmodified forms ([M + 3H]3+ = m/z 622.30) (lower). For both hydroxylated spectra, a −2 Da mass shift was commonly observed on fragment ions containing hydroxyhistidine, which is consistent with hydroxylation (+16 Da) followed by dehydration (−18 Da). Because there was no evidence for a −2 Da shift on the precursor ions it is likely that during the collision-induced dissociation process in the MS/MS analyses, the hydroxylated His residue undergoes dehydration to form the conjugated α,β-dehydrohistidine product. Note also there was no evidence for formation of the dehydrohistidine in the NMR analyses (Fig. 4).
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.