Cloning and expression of human deoxyhypusine synthase cDNA. Structure-function studies with the recombinant enzyme and mutant proteins.
Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculated molecular mass of 40,970 Da) and a shorter cDNA clone that would potentially encode a protein with an internal deletion of 56 amino acids (Asp262-Ser317) were isolated. The deduced amino acid sequence of the human enzyme shows a high degree of identity to that of yeast deoxyhypusine synthase and to the known sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine synthesis. Variant human recombinant proteins with (i) a truncation of 48 or 97 NH2-terminal amino acids, (ii) a truncation of 39 COOH-terminal amino acids, or (iii) an internal deletion (Asp262-Ser317) were inactive. A chimeric protein consisting of the complete human sequence and 16 amino acids of the yeast sequence (Gln197-Asn212, not present in the human enzyme) inserted between Glu193 and Gln194 exhibited moderate activity.
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Enzyme Chemistry Section, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4330, USA.
Wolff
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Park
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase