Saccharomyces cerevisiae GNA1, an Essential Gene Encoding a Novel Acetyltransferase Involved in UDP-N-acetylglucosamine Synthesis

Abstract

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 ofAGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1(glucosamine-6-phosphateacetyltransferase). 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 ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 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 Phe142 and Tyr143 residues are essential for the catalysis. 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 ofAGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1(glucosamine-6-phosphateacetyltransferase). 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 ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 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 Phe142 and Tyr143 residues are essential for the catalysis. open reading frame glutathione S-transferase. Acetyltransferases catalyze the acetylation of their specific substrates using the cofactor acetyl-CoA (Ac-CoA). These enzymes play important roles in a wide variety of biological processes, including metabolism, chromatin structure, gene expression, and cell cycle (1Imhof A. Yang X.-J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 2Meyer U.A. Zanger U.M. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 269-296Crossref PubMed Scopus (492) Google Scholar, 3Wade P.A. Pruss D. Wolffe A.P. Trends Biol. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (410) Google Scholar). At least one acetyltransferase is necessary for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential metabolite both in prokaryotes and eukaryotes. In bacteria, UDP-GlcNAc is a major cytoplasmic precursor of the cell wall peptide glycan, and it is also used as the disaccharide moiety of lipid A (4Holtje J.V. Schwartz U. Nanninga N. Molecular Cytology of Escherichia coli. Academic Press, Inc., New York1985: 77-119Google Scholar, 5Park J.T. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurinum: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1987: 663-671Google Scholar, 6Raetz C.R.H. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurinum: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1987: 498-503Google Scholar). In eukaryotes, it serves as the substrate of chitin synthase and is also utilized for the GlcNAc moiety of N-linked glycosylation as well as the glycosylphosphatidylinositol-anchor of cellular proteins (7Cabib E. Roberts R. Bowers B. Annu. Rev. Biochem. 1982; 51: 763-793Crossref PubMed Scopus (270) Google Scholar, 8Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (440) Google Scholar). In Escherichia coli, biosynthesis of UDP-GlcNAc starts from the conversion of fructose 6-phosphate (Fru-6-P) into glucosamine 6-phosphate (GlcN-6-P) by glutamine:Fru-6-P amidotransferase. Then, GlcN-6-P is converted into glucosamine 1-phosphate (GlcN-1-P) by phosphoglucosamine (phospho-GlcN) mutase. Acetylation occurs on GlcN-1-P, and the resulting N-acetylglucosamine 1-phosphate (GlcNAc-1-P) is uridylated by UDP-GlcNAc pyrophosphorylase (9Dobrogosz W.J. J. Bacteriol. 1968; 95: 578-584Crossref PubMed Google Scholar, 10White R.J. Biochem. J. 1968; 106: 847-858Crossref PubMed Scopus (116) Google Scholar, 11Freese E.B. Cole R.M. Klofat W. Freese E. J. Bacteriol. 1970; 101: 1046-1062Crossref PubMed Google Scholar). TheGlmS and GlmM genes of E. coli code for glutamine:Fru-6-P amidotransferase (12Wu H.C. Wu T.C. J. Bacteriol. 1971; 105: 455-466Crossref PubMed Google Scholar, 13Walker J.E. Gay N.J. Saraste M. Eberle A.N. Biochem. J. 1984; 224: 799-815Crossref PubMed Scopus (107) Google Scholar, 14Dutka-Malen S. Mazodier P. Badet B. Biochimie (Paris). 1988; 70: 287-290Crossref PubMed Scopus (51) Google Scholar) and phospho-GlcN mutase (15Mengin-Lecreulx D. van Heijenoort J. J. Biol. Chem. 1996; 271: 32-39Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), respectively. In addition, the product of the E. coli GlmU gene is a bifunctional protein having both GlcN-1-P acetyltransferase and UDP-GlcNAc pyrophosphorylase activities (16Mengin-Lecreulx D. van Heijenoort J. J. Bacteriol. 1993; 175: 6150-6157Crossref PubMed Google Scholar, 17Mengin-Lecreulx D. van Heijenoort J. J. Bacteriol. 1994; 176: 5788-5795Crossref PubMed Scopus (166) Google Scholar). In Saccharomyces cerevisiae, GFA1 andAGM1 are responsible for the synthesis of GlcN-6-P from Fru-6-P and for the interconversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, respectively (18Watzele G. Tanner W. J. Biol. Chem. 1989; 264: 8753-8758Abstract Full Text PDF PubMed Google Scholar, 19Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). As Agm1p utilizes phospho-N-acetylglucosamine (phospho-GlcNAc) as the substrate, it has been assumed that acetyl transfer precedes the mutase reaction, and, therefore, GlcN-6-P would serve as the recipient of the acetyl moiety (19Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). In our previous paper, we reported that the yeast UAP1 gene encodes UDP-GlcNAc pryophosphorylase and demonstrated that, unlike the product of the E. coli GlmUgene, phospho-GlcN acetyltransferase activity is not intrinsic in the yeast Uap1 protein (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The human UDP-GlcNAc pyrophosphorylase was also identified and shown to be highly related to the yeast enzyme. Curiously, the human enzyme turned out to be identical to the antigen of male infertility patients (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 21Diekman A.B. Goldberg E. Biol. Reprod. 1994; 50: 1087-1093Crossref PubMed Scopus (39) Google Scholar, 22Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Furthermore, the human enzyme recognizes both UDP-GlcNAc and UDP-GalNAc, but the deletion of 17 amino acids in the C-terminal region, which occurs physiologically by alternative splicing, alters its substrate specificity (22Wang-Gillam A. Pastuszak I. Elbein A.D. J. Biol. Chem. 1998; 273: 27055-27057Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In this paper, we report the S. cerevisiae gene for phospho-GlcN acetyltransferase. The enzyme utilizes GlcN-6-P as the substrate, and its deletion is lethal, leading to a morphology similar to those observed in UAP1- and AGM1-deficient cells. The important residues for the catalysis are proposed on the basis of mutation analysis. The entire open reading frame (ORF)1 ofScGNA1 (YFL107C) was amplified by polymerase chain reaction with the S. cerevisiae genomic DNA extracted from strain A451 (MATα can1 aro7 can1 leu2 trp1 ura3) as a template. It was then cloned at the XbaI site of pUC18 or pYEUra3 (Toyobo), generating pUC-ScGNA1 and pYEU-ScGNA1, respectively. Primers used for polymerase chain reaction were 5′-AAGGATCCAGCTTACCCGATGGATTTTATATA-3′ and 5′-AAGAATTCCTATTTTCTAATTTGCATTTCCAC-3′. The Candida albicanshomolog of ScGNA1 was cloned by screening a C. albicans genomic DNA library as a probe using the 0.2-kilobase fragment of the C. albicans genomic DNA, whose sequence was available in the C. albicans data base and was found to be related to YFL017C. The filters were hybridized and washed under stringent conditions (20 mm sodium phosphate (pH 7.2), 5 × SSC (1 × SSC contains 150 mm NaCl and 15 mm sodium citrate), 5 × Denhardt's solution, 0.1% SDS, 25% formamide at 42 °C for hybridization; 0.1 × SSC and 0.1% SDS at 50 °C for washing). After the third screening, DNA was extracted from bacterial cells that were strongly hybridized with the probe DNA, and the insert DNA was cloned between theBamHI and SalI sites of pUC19 for further plasmid construction. Radiolabeling of the probe DNA was performed by the random priming method using [α-32P]dCTP (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.31-13.77Google Scholar), and DNA sequencing was carried out as described elsewhere (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.31-13.77Google Scholar). Construction of the C. albicans genomic DNA library was already reported (24Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar). The coding regions of ScGNA1 and CaGNA1 were cloned between the BamHI and EcoRI sites of pGEX-2T (25Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar), and the resulting plasmids were transfected into E. coli JM109 to express the recombinant yeast proteins as a fusion with glutathione S-transferase (GST). Induction and expression of the recombinant Gna1 proteins was carried out with isopropyl-β-d-thiogalactopyranoside as described (24Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar,25Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). At 4 h after the addition of isopropyl-β-d-thiogalactopyranoside, the bacterial cells were harvested, suspended in a buffer containing 20 mmTris-HCl (pH 7.5), 0.5 mm EDTA, 10%(v/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, and lysed by sonication. After cell debris was removed by centrifugation at 15,000 ×g at 4 °C for 30 min, GST-Gna1 fusion proteins were purified by glutathione-Sepharose CL-4B column chromatography, as described (25Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar), and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The primers used for amplifying the CaGNA1 ORF were 5′-AAGGATCCTTACCACAAGGTTATACATTCAGA-3′ and 5′-AAGAATTCCTAGAATCTACATACCATTTCAAC-3′. Because CoA reacts with 2-nitrobenzoic acid and releases 4-nitrothiophenolate (26Gehring A.M. Lees W.J. Mindiola D.J. Walsh C.T. Brown E.D. Biochemistry. 1996; 35: 579-585Crossref PubMed Scopus (86) Google Scholar,27Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; PubMed Scopus Google Scholar), an for phospho-GlcN acetyltransferase was performed in 50 of a reaction containing 50 mm (pH 7.5), 5 mm glycerol, and 0.1 of the recombinant fusion After at 30 °C for the the reaction was by 50 of a containing 50 mmTris-HCl (pH and m and then 50 of a containing 50 mm (pH 7.5), 1 mm EDTA, and 20 2-nitrobenzoic The of CoA produced by the Gna1 protein were from that of 4-nitrothiophenolate by the at The UDP-GlcNAc synthesis was carried out in a 20 reaction containing 50 mm (pH 5 mm glycerol, 0.1 1 × and 0.1 of and Uap1 proteins at 30 °C for of the reaction were and were by in a that was by of of and of in of K. E. Biochem. PubMed Scopus Google Scholar). The were by The entire ORF ofScGNA1 was cloned between the BamHI and SalI sites of pUC18 and pYEUra3 of the generating pUC-ScGNA1 and pYEU-ScGNA1, respectively. Then, the of ORF in pUC-ScGNA1 was and by generating The strain was with pYEU-ScGNA1, and were further transfected with that been and The resulting which in but not in were and used as strain leu2 The S. cerevisiae leu2 and leu2 were already described in the previous (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). the of to the entire ORF of CaGNA1 was cloned at the BamHI site of the of (24Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar). the of CaGNA1 from this plasmid was under the of the The resulting plasmid was transfected into cells. After the cells were in the of they were to containing and further for A of the an alanine for Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, or Gly147 were by the method as described T. T. Y. M. Gene (Amst.). PubMed Scopus Google Scholar) with The entire ORF of the ScGNA1 gene was cloned between the BamHI and EcoRI sites of using EcoRI and hybridized with containing the The resulting ScGNA1 genes were from the and with which been with BamHI the were by sequencing the we demonstrated that unlike the bacterial the UDP-GlcNAc pyrophosphorylase is a protein with phospho-GlcN acetyltransferase activity (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). When the S. cerevisiae data we on encodes a protein with to the we an S. cerevisiae strain in which the YFL017C gene was but of whose was under the of the were The resulting strain on but on that YFL017C is an essential When on of the cells and then This morphology was similar to that caused by the depletion of or UAP1 suggesting that YFL017C is in the synthesis of Although the product of YFL017C a sequence to the C-terminal phospho-GlcN acetyltransferase of the gene product was in this is a gene for an essential as UDP-GlcNAc it be in other A of the C. albicans data base a sequence that sequence cerevisiae screening albicans genomic DNA library with the DNA fragment of the sequence as a we were to and sequence the C. albicans of The of ScGNA1 are related to other sequence the entire In addition, under the of CaGNA1 an S. cerevisiae not that the CaGNA1 Furthermore, a of the data that S. and C. proteins which a of sequence with ScGna1p the entire protein between ScGna1p and the product and between ScGna1p and the we to these genes as S. and C. respectively. Although the and to be the results strongly the that acetyltransferase necessary for UDP-GlcNAc synthesis. In UDP-GlcNAc is a acetyltransferase that is the acetylation of we specifies phospho-GlcN acetyltransferase. and proteins were in E. coli as a fusion with and then purified by column When incubated with GlcN-6-P and and produced whereas not The of CoA from a 5 and GlcN-6-P was utilized as the substrate Although of CoA were produced of GlcN-1-P were the K m value for GlcN-1-P was that for GlcN-6-P mm for GlcN-1-P for CoA was observed 1-phosphate was used as the substrate of the was also to alanine as a K m and of the and enzymes for were from the of CoA produced from The K m for GlcN-6-P are also in the not A was also to alanine as a in a The K m and of the and enzymes for were from the of CoA produced from The K m for GlcN-6-P are also in the not we demonstrated that with produced UDP-GlcNAc from (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). the acetylation of GlcN-6-P and UDP-GlcNAc would be from GlcN-6-P in the of and As and but not produced from the yeast Uap1p, and were to the reaction A to was also observed in This is to a of in the it in the of the enzyme of UDP-GlcNAc from GlcN-6-P acetyl-CoA, and that using Although not GlcN-1-P as the substrate in the acetyltransferase was from GlcN-1-P Uap1p, and were mutase an essential for the synthesis of UDP-GlcNAc from was in the of Agm1p This that Agm1p recognizes both phospho-GlcN and as the Agm1p GlcN-1-P into the substrate for and then which was from GlcN-6-P by we that encodes GlcN-6-P and for the of UDP-GlcNAc from 0.1 of the purified was incubated with 0.1 of and 0.1 in the or of 0.1 of GlcN-1-P, and After at 30 °C for min, the reaction were by and by The of and under are A of been identified in When the amino acid sequence of ScGna1p was compared with and putative acetyltransferases, we found that the bacterial GlmU protein is not highly related to of the amino acids in the regions of ScGna1p are highly These designated and the amino acid from to and from to respectively. the of these for we the highly amino acids in these regions Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 to As a a was also converted to After the the enzymes were as a fusion with and purified by column As shown in alanine for Val102, Gly112, Leu115, Ile116, and Gly147 the In those for Tyr143 severely the Furthermore, the activity of Y143A was too low to perform kinetics, alanine for the amino acid the K m for and GlcN-6-P by and respectively The activities of and were also to a these displayed of the to the cofactor and the substrate. of increased the K m value for GlcN-6-P by whereas that of the K m value for by on ScGna1p activity of alanine for the amino The and the proteins an alanine for of the amino acids that are highly in in were as a fusion with and purified with glutathione-Sepharose of the and the proteins were on a gel and with The of the protein and are in and by respectively. 0.1 of the purified and the proteins were incubated with of GlcN-6-P and at 30 °C for min, and the of the CoA that the enzyme activities were as described in The activities of the enzymes are as a of that of the In human it was demonstrated that the amino acid sequence at the of is for the and that and the in this are essential for the enzyme activity J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the as a H. Biochem. J. 1996; PubMed Scopus Google Scholar). In our sequence and of to and of respectively As the and other amino acids in the are not highly in in and we of ScGna1p to the of the mutation of was not as as those of Phe142 and Tyr143, the activity and the of were significantly that of the and m value for was that of the enzyme these it was that Phe142 and Tyr143 of ScGna1p are essential residues for the and that Ile97, Glu98, and in the and the of the cofactor and the substrate. In this paper, we identified the yeast as the gene for phospho-GlcN and demonstrated that unlike the in bacteria, yeast UDP-GlcNAc pyrophosphorylase and phospho-GlcN acetyltransferase are encoded by distinct and essential genes (18Watzele G. Tanner W. J. Biol. Chem. 1989; 264: 8753-8758Abstract Full Text PDF PubMed Google Scholar, 19Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar, T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), of the UDP-GlcNAc synthesis is essential for the of yeast cells. In addition, yeast has one of amino that is is in yeast cells. is to be the amino acetyltransferase in yeast cells. ScGna1p a K m value for compared with those of other for and for J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Biochem. J. 1996; PubMed Scopus Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This is also for the E. coli phospho-GlcN acetyltransferase (26Gehring A.M. Lees W.J. Mindiola D.J. Walsh C.T. Brown E.D. Biochemistry. 1996; 35: 579-585Crossref PubMed Scopus (86) Google Scholar). low to is of phospho-GlcN The K of ScGna1p for GlcN-6-P was also The enzyme displayed a substrate specificity to the K for GlcN-1-P was that for ScGna1p produced UDP-GlcNAc from GlcN-1-P in the of Uap1p, and Because not as the substrate (20Mio T. Yabe T. Arisawa M. Yamada-Okabe H. J. Biol. Chem. 1998; 273: 14392-14397Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), the results in this indicate that Agm1p has a substrate specificity and recognizes phospho-GlcN as In not but also be the substrate of Agm1p (19Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). Although which is not in the yeast data a of the that the amino acid sequence of ScGna1p is identical to that of reported R. 1996; PubMed Scopus Google Scholar). was as the gene that the of the S. cells R. 1996; PubMed Scopus Google Scholar). of results in The cells displayed cell increased to the structure, and of Furthermore, by cell it was demonstrated that is for in the cell as from of DNA and R. 1996; PubMed Scopus Google Scholar). these a between UDP-GlcNAc synthesis and cell cycle As UDP-GlcNAc is used as the cell wall precursor in we has as whose acetylation is essential for gene expression and cell cycle acetyltransferase activity was in the recombinant R. 1996; PubMed Scopus Google Scholar) cloned the by the of an S. cerevisiae Although the amino acid sequence of the C-terminal of S. is identical to that of the identified has amino acids at the R. 1996; PubMed Scopus Google Scholar). Because we not the DNA sequence to in the S. data the of the amino acids in be to a S. of the that ScGna1p contains amino acids are highly and putative the mutation we found that Phe142 and Tyr143 of ScGna1p are amino acids for the reaction and that Ile97, Glu98, and also play important roles in the of the cofactor and the substrate. As at the of and the of been to be the site of the enzyme J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Biochem. J. 1996; PubMed Scopus Google Scholar). Although of is converted to in the yeast Gna1 our results indicate that of ScGna1p is also in the In alanine for the highly Gly112, which to the third in the on the activity of and other in the are not highly in ScGna1p and its the of the yeast acetyltransferase and both of which to the were and the was demonstrated to an important of the R. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. Y. A. Nakatani Y. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). our results that Phe142 and Tyr143 in of play important roles in the Because Phe142 Tyr143 is in of it is to the roles of these amino acids in catalysis. Phe142 and Tyr143 in are in the with the base of S. for data base and sequence Y. for with the and S. B. for reading the