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
Mentions: Previously, we have reported that various human ARD-containing proteins undergo hydroxylation at conserved Asn residues [5,6,8,9]. One of the most highly modified is tankyrase-2, which undergoes FIH-catalysed hydroxylation at eight Asn residues . Alignment of the tankyrase-2 ARD revealed two His residues (His 238 and His 553) embedded within the FIH hydroxylation consensus comprising an ‘LxxxxxDVH’ motif at positions analogous to proven FIH-catalysed Asn hydroxylation sites (Fig. 1A) . The positioning of these His residues within the hydroxylation consensus, coupled with the structural similarity between Asn and His residues, raised the interesting possibility that His 238 and/or His 553 of tankyrase-2 might also be hydroxylated by FIH. To test this hypothesis, we prepared synthetic 21-residue peptides encompassing the two His residues of interest and tested them as FIH substrates. Significantly, both peptides (TNKS2 223–243 and TNKS2 538–558) displayed a clear +16 Da mass increment after reaction with FIH under standard assay conditions (Fig. 1B). MS/MS analyses of the modified TNKS2 538–558 peptide after tryptic digestion assigned the site of hydroxylation to that corresponding to His 553 in the tankyrase-2 ARD (Fig. S1).
Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.