The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases GENE CLONING, PROTEIN EXPRESSION, AND CATALYTIC MECHANISM
作者： Toshiyuki Mio , Tomio Yabe , Mikio Arisawa , Hisafumi Yamada-Okabe
关键词: Molecular biology 、 Biochemistry 、 cDNA library 、 Complementary DNA 、 Sequence analysis 、 Saccharomyces cerevisiae 、 Genomic library 、 Biology 、 Molecular cloning 、 Peptide sequence 、 Gene
摘要: A search of the yeast data base for a protein homologous to Escherichia coliUDP-N-acetylglucosamine pyrophosphorylase yieldedUAP1 (UDP-N-acetylglucosaminepyrophosphorylase), Saccharomyces cerevisiae gene UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening C. genomic library testis cDNA library, respectively. Sequence analysis revealed that UAP1 was identical previously reported AGX1. null mutation S. (ScUAP1) lethal, when expressed under control ScUAP1 promoter, bothC. Homo sapiens UAP1(CaUAP1 HsUAP1) rescued theScUAP1-deficient cells. All recombinant ScUap1p, CaUap1p, HsUap1p possessed activitiesin vitro. Uap1p utilizedN-acetylglucosamine-1-phosphate as substrate, together with Agm1p, it produced from N-acetylglucosamine-6-phosphate. These results demonstrate genes indeed specify eukaryotic UDP-GlcNAc phosphomutase reaction precedes uridyltransfer. comparison other UDP-sugar pyrophosphorylases amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, Lys123 ScUap1p are highly conserved in date. Among these acids, alanine substitution or severely diminished activity, suggesting possible catalytic residues enzyme.
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