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J. Biol. Chem., Vol. 282, Issue 11, 8300-8308, March 16, 2007

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Specificity of the Deoxyhypusine Hydroxylase-Eukaryotic Translation Initiation Factor (eIF5A) Interaction

IDENTIFICATION OF AMINO ACID RESIDUES OF THE ENZYME REQUIRED FOR BINDING OF ITS SUBSTRATE, DEOXYHYPUSINE-CONTAINING eIF5A^*

From the Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (N-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, 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 (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu⁵⁷, Glu⁹⁰, Glu²⁰⁸, Glu²⁴¹, Gly⁶³, or Gly²¹⁴ caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by -carboxyl groups of Glu⁵⁷ and Glu²⁰⁸ at the DOHH active site.

Received for publication, August 7, 2006 , and in revised form, January 8, 2007.

^* This work was supported by the Intramural Research Program of the National Institutes of Health (NIDCR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Current address: Medical Research Center for Neural Dysfunction, Dept. of Biochemistry, School of Medicine and Institute of Health Sciences, Gyeongsang National University, 92 Chilam-Dong, Jinju 660-751, Korea.

³ To whom correspondence should be addressed: Bldg. 30, Rm. 211, OPCB, NIDCR, National Institutes of Health, Bethesda MD 20892-4340. Tel.: 301-496-5056; Fax: 301-402-0823; E-mail: mhpark{at}nih.gov.

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