Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized, Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFLO17C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine B-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine B-phosphate, These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGnalp was compared with those of other acetyltransferases, Ile(97), Glu(98), Val(102), Gly(112), Leu(115), Ile(116), Phe(142), Tyr(143), and Gly(147) were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe(142) or Tyr(143) enzymes was severely diminished, Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased K-m value for acetyl-CoA, suggesting that the Phe(142) and Tyr(143) residues are essential for the catalysis.