Substrate specificity of deoxyhypusine hydroxylase

The eukaryotic initiation factor 5A (eIF5A) is the only cellular protein containing the unique amino acid hypusine [N ε ‐(4‐amino‐2‐hydroxybutyl)lysine]. Hypusine is formed posttranslationally by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The specificity of interaction between eIF5A and DOHH was investigated using human eIF5A and human recombinant DOHH. DOHH displayed a strong preference for binding its natural substrate, the deoxyhypusine‐containing form of eIF5A, [eIF5A(Dhp)], over the eIF5A precursor or the hypusine‐containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>aa20‐90) is required for effective modification by DOHH. The amino acid residues of DOHH that are critical for binding of the substrate were identified by site‐directed mutagenesis. Alanine substitutions at Glu57, Glu90, Glu208, Glu241, Gly63 and Gly214 caused impairment in eIF5A(Dhp) binding and activity, suggesting the importance of these amino acid residues in the eIF5A(Dhp)/DOHH interaction. These findings support a mode of DOHH‐eIF5A binding in which the deoxyhypusine side chain of the substrate protein is primarily anchored by Glu 57 and Glu 208 of the DOHH active site. This research was supported by the Intramural Program of the National Institutes of Health (NIDCR).