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: Having established that His-containing peptides are FIH substrates in vitro, we then investigated whether tankyrase-2 might be subject to FIH-catalysed His hydroxylation in cells. To address this, we transfected plasmids encoding full-length FLAG-tagged tankyrase-2 and FIH into 293T cells, immunopurified the material by FLAG affinity and subjected it to trypsinolysis and MS/MS analysis. Peptides containing both His residues were observed, and MS/MS sequencing assigned hydroxylation at His 238 and His 553 (Fig. 2). As previously observed for hydroxyasparagine-containing peptides , under our HPLC conditions, the hydroxyhistidine modification had minimal effect on the peptide chromatographic properties and the hydroxylated and nonhydroxylated peptides coeluted (data not shown). The exact masses and retention times of the His-containing peptides were subsequently used to assign the hydroxylated and nonhydroxylated peptides studied by LC/MS analyses.
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.