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 th e formation of a deoxyhypusine residue on the eukaryotic initiation fa ctor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from s permidine to the epsilon-amino group of a unique lysine residue. We ha ve recently cloned and characterized the Neurospora crassa deoxyhypusi ne synthase cDNA using a reverse genetics approach. A GenBank search s howed that a stretch of the deduced amino acid sequence (96 amino acid s) of Neurospora deoxyhypusine synthase matches a short human expresse d sequence tag (EST), Z25337, with greater than 70% amino acid identit y. Gene-specific primers based on this EST were used together with uni versal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA l ibrary. The 1219 bp and 1078 bp sequences, each containing an open rea ding frame, encode polypeptides of respectively 368 and 321 amino acid s. The short sequence is identical to the long one except that it is m issing a stretch of 47 amino acids spanning residues 261-307. The 368- amino-acid sequence of human deoxyhypusine synthase shares a high degr ee of identity (> 50%) and similarity (> 60%) with that of the Neurosp ora and yeast deoxyhypusine synthases. After cloning into an expressio n vector, the 368-amino-acid recombinant protein exhibits high deoxyhy pusine synthase activity. In contrast, the 321-amino-acid recombinant protein shows no detectable activity.