Molecular cloning and functional expression of human deoxyhypusine synthase cDNA based on expressed sequence tag information.
Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyses the formation of a deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from spermidine to the epsilon-amino group of a unique lysine residue. We have recently cloned and characterized the Neurospora crassa deoxyhypusine synthase cDNA using a reverse genetics approach. A GenBank search showed that a stretch of the deduced amino acid sequence (96 amino acids) of Neurospora deoxyhypusine synthase matches a short human expressed sequence tag (EST), Z25337, with greater than 70% amino acid identity. Gene-specific primers based on this EST were used together with universal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA library. The 1219 bp and 1078 bp sequences, each containing an open reading frame, encode polypeptides of respectively 368 and 321 amino acids. The short sequence is identical to the long one except that it is missing a stretch of 47 amino acids spanning residues 261-307. The 368-amino-acid sequence of human deoxyhypusine synthase shares a high degree of identity ( > 50%) and similarity ( > 60%) with that of the Neurospora and yeast deoxyhypusine synthases. After cloning into an expression vector, the 368-amino-acid recombinant protein exhibits high deoxyhypusine synthase activity. In contrast, the 321-amino-acid recombinant protein shows no detectable activity.
Yan
Y P
YP
Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939, USA.
Tao
Y
Y
Chen
K Y
KY
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase