IDENTIFICATION OF YHR068W IN SACCHAROMYCES-CEREVISIAE CHROMOSOME-VIIIAS A GENE FOR DEOXYHYPUSINE SYNTHASE - EXPRESSION AND CHARACTERIZATION OF THE ENZYME

Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the f irst step in hypusine biosynthesis. Amino acid sequences of five trypt ic peptides from rat deoxyhypusine synthase were found to match partia lly the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061). In or der to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase, a 1.17-kilobase pair cDNA with an identical n ucleotide sequence to that of the YR068w coding region was obtained fr om S. cerevisiae cDNA by polymerase chain reaction and was expressed i n Escherichia coli B strain BL21(DE3). The recombinant protein was fou nd mostly in the E. coli cytosol fraction and comprised similar to 20% of the total soluble protein. The purified form of the expressed prot ein effectively catalyzed the formation of deoxyhypusine in yeast eIF- 5A precursors as well as in human precursor and in those from Chinese hamster ovary cells. The molecular mass of the enzyme was estimated to be 172,000 a 4,300 Da by equilibrium centrifugation. The mass of its polypeptide subunit was determined to be similar to 43,000 Da, in clos e agreement with that calculated for the coding region of the YHR068w gene. These findings show that this gene is a coding sequence for yeas t deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.