Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs.
Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.
Huang
Jenq-Kuen
JK
Department of Chemistry, Western Illinois University, 1 University Circle, Macomb, IL 61455-1390, USA.
Cui
Yalun
Y
Chen
Chieh-Hua
CH
Clampitt
Denae
D
Lin
Chi-Tsai
CT
Wen
Lisa
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D000097574:Eukaryotic Translation Initiation Factor 5A
D018076:DNA, Complementary; D006899:Mixed Function Oxygenases; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C047991:deoxyhypusine hydroxylase