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Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

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FIH catalysed His-hydroxylation occurs at the β-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was produced by incubation with FIH under standard assay conditions (hydroxylated to ∼ 75% as assessed by MALDI-TOF analyses), LC-MS purified and analysed by NMR spectroscopy. (B) 1H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O. The resonances at 4.87 and 5.50 ppm, which are absent in the 1H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the α- and β-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D 1H–1H COSY spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O indicating the 1H–1H correlation between resonances arising from the α- and β-hydrogens of the hydroxylated His residue.
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fig04: FIH catalysed His-hydroxylation occurs at the β-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was produced by incubation with FIH under standard assay conditions (hydroxylated to ∼ 75% as assessed by MALDI-TOF analyses), LC-MS purified and analysed by NMR spectroscopy. (B) 1H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O. The resonances at 4.87 and 5.50 ppm, which are absent in the 1H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the α- and β-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D 1H–1H COSY spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O indicating the 1H–1H correlation between resonances arising from the α- and β-hydrogens of the hydroxylated His residue.

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).


Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

FIH catalysed His-hydroxylation occurs at the β-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was produced by incubation with FIH under standard assay conditions (hydroxylated to ∼ 75% as assessed by MALDI-TOF analyses), LC-MS purified and analysed by NMR spectroscopy. (B) 1H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O. The resonances at 4.87 and 5.50 ppm, which are absent in the 1H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the α- and β-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D 1H–1H COSY spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O indicating the 1H–1H correlation between resonances arising from the α- and β-hydrogens of the hydroxylated His residue.
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Related In: Results  -  Collection

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fig04: FIH catalysed His-hydroxylation occurs at the β-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was produced by incubation with FIH under standard assay conditions (hydroxylated to ∼ 75% as assessed by MALDI-TOF analyses), LC-MS purified and analysed by NMR spectroscopy. (B) 1H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O. The resonances at 4.87 and 5.50 ppm, which are absent in the 1H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the α- and β-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D 1H–1H COSY spectrum of the hydroxylated TNKS2 538–558 peptide in 2H2O indicating the 1H–1H correlation between resonances arising from the α- and β-hydrogens of the hydroxylated His residue.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

Show MeSH
Related in: MedlinePlus