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

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferas e was characterized, Disruption of YFL017C was lethal, leading to a morphol ogy similar to those caused by the depletion of AGM1 or UAP1, the genes enc oding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyroph osphorylase, 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 gluc osamine B-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine B-pho sphate, These results indicate that YFL017C specifies glucosamine-6-phospha te acetyltransferase; therefore, the gene was designated GNA1 (glucosamine- 6-phosphate acetyltransferase). In addition, whereas bacterial phosphogluco samine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase acti vities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGnalp was compared with th ose 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 co nserved. When alanine was substituted for these amino acids, the enzyme act ivity for the substituted Phe(142) or Tyr(143) enzymes was severely diminis hed, Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased K-m value for acetyl-CoA, suggesting th at the Phe(142) and Tyr(143) residues are essential for the catalysis.