THE EUKARYOTIC UDP-N-ACETYLGLUCOSAMINE PYROPHOSPHORYLASES - GENE CLONING, PROTEIN EXPRESSION, AND CATALYTIC MECHANISM

A search of the yeast data base for a protein homologous to Escherichi a coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-a cetylglucosamine pyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans a nd human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analys is revealed that the human UAP1 cDNA was identical to previously repor ted AGX1., A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, both C. albicans and Homo sapiens UAP1 (CaUAP1 and HsUAP1) rescued the ScU AP1-deficient S., cerevisiae cells. All the recombinant ScUap1p, CaUap 1p, and HsUap1p possessed UDP-N-acelylglucosamine pyrophosphorylase ac tivities in vitro. The yeast Uap1p utilized N-acetylglucosamirne-1-pho sphate as the substrate, and together with Agm1p, it produced UDP-N-ac etylglucosamine from N-acetylglucosamine-6-phosphate. These results de monstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyr ophosphorylase and that phosphomutase reaction precedes uridyltransfer ., Sequence comparison with other UDP-sugar pyrophosphorylases reveale d that amino acid residues, Gly(112) Gly(114), Thr(115), Arg(116), Pro (122), and Lys(123) of ScUap1p are highly conserved in UDP-sugar pyrop hosphorylases reported to date, Among these amino acids, alanine subst itution for Gly(112), Arg(116), or Lys(123) severely diminished the ac tivity, suggesting that Gly(112), Arg(116), or Lys(123) are possible c atalytic residues of the enzyme.