Molecular cloning and functional expression of Neurospora deoxyhypusine synthase cDNA and identification of yeast deoxyhypusine synthase cDNA.
Deoxyhypusine synthase catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor using spermidine as the substrate. We have purified deoxyhypusine synthase from Neurospora crassa to apparent homogeneity (Tao, Y., and Chen, K. Y. (1995) J. Biol. Chem. 270, 383-386). We have now cloned and characterized the deoxyhypusine synthase cDNA using a reverse genetic approach. Conceptual translation of the nucleotide sequence of the cloned 1258-base pair cDNA revealed an open reading frame containing 353 amino acids with a predicted M(r) of 38,985. The deoxyhypusine synthase cDNA was subcloned into the expression vector pQE60 to produce a 40,000-dalton recombinant protein on SDS-PAGE which exhibited deoxyhypusine synthase activity. A GenBank search showed that the Neurospora deoxyhypusine synthase cDNA possessed significant sequence homology to a previously uncharacterized yeast sequence (accession number U00061 (1994)). The yeast sequence encodes a protein of 387 amino acids that shows 69% of total amino acid identity and 80% of total amino acid similarity to the Neurospora enzyme. Sequence alignment and hydropathy analysis suggest that the yeast sequence represents deoxyhypusine synthase.
Tao
Y
Y
Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939, USA.
Chen
K Y
KY
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology* / Q000235:genetics*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase