The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases

A search of the yeast data base for a protein homologous to Escherichia coliUDP-N-acetylglucosamine pyrophosphorylase yieldedUAP1 (UDP-N-acetylglucosaminepyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, bothC. albicans and Homo sapiens UAP1(CaUAP1 and HsUAP1) rescued theScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activitiesin vitro. The yeast Uap1p utilizedN-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 of ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported to date. Among these amino acids, alanine substitution for Gly112, Arg116, or Lys123 severely diminished the activity, suggesting that Gly112, Arg116, or Lys123 are possible catalytic residues of the enzyme. A search of the yeast data base for a protein homologous to Escherichia coliUDP-N-acetylglucosamine pyrophosphorylase yieldedUAP1 (UDP-N-acetylglucosaminepyrophosphorylase), the Saccharomyces cerevisiae gene for UDP-N-acetylglucosamine pyrophosphorylase. The Candida albicans and human homologs were also cloned by screening a C. albicans genomic library and a human testis cDNA library, respectively. Sequence analysis revealed that the human UAP1 cDNA was identical to previously reported AGX1. A null mutation of the S. cerevisiae UAP1 (ScUAP1) gene was lethal, and when expressed under the control of ScUAP1 promoter, bothC. albicans and Homo sapiens UAP1(CaUAP1 and HsUAP1) rescued theScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activitiesin vitro. The yeast Uap1p utilizedN-acetylglucosamine-1-phosphate as the substrate, and together with Agm1p, it produced UDP-N-acetylglucosamine from N-acetylglucosamine-6-phosphate. These results demonstrate that the UAP1 genes indeed specify eukaryotic UDP-GlcNAc pyrophosphorylase and that phosphomutase reaction precedes uridyltransfer. Sequence comparison with other UDP-sugar pyrophosphorylases revealed that amino acid residues, Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 of ScUap1p are highly conserved in UDP-sugar pyrophosphorylases reported to date. Among these amino acids, alanine substitution for Gly112, Arg116, or Lys123 severely diminished the activity, suggesting that Gly112, Arg116, or Lys123 are possible catalytic residues of the enzyme. UDP-N-acetylglucosamine (UDP-GlcNAc 1The abbreviations used are: UDP-GlcNAc, UDP-N-acetylglucosamine; ORF, open reading frame; GST, glutathione S-transferase; GlcN, glucosamine; Fru-6-P, fructose-6-phosphate; GlcN-6-P, glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; Man-6-P, mannose-6-phosphate; Man-1-P, mannose-1-phosphate; Gal-1-P, galactose-1-phosphate; Glc-1-P, glucose-1-phosphate; TLC, thin layer chromatography. ) is a ubiquitous and essential metabolite and plays important roles in several metabolic processes. In bacteria, it is known as a major cytoplasmic precursor of cell wall peptide glycan and the disaccharide moiety of lipid A (1Holtje J.V. Schwartz U. Nanninga N. Molecular Cytology of Escherichia coli. Academic Press, Inc., New York1985: 77-119Google Scholar, 2Park 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, 3Raetz 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, whose product is shown to be essential for fungal cell wall (4Cabib E. Roberts R. Bowers B. Annu. Rev. Biochem. 1982; 51: 763-793Crossref PubMed Scopus (270) Google Scholar). It is also used in the GlcNAc moiety of N-linked glycosylation and the GPI-anchor of cellular proteins (5Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (440) Google Scholar). Biosynthesis of UDP-GlcNAc has been extensively studied in bacteria, and it requires the following enzymatic reactions: i) conversion of fructose-6-phosphate (Fru-6-P) into glucosamine-6-phosphate (GlcN-6-P) by glutamine:Fru-6-P amidotransferase; ii) conversion of GlcN-6-P into glucosamine-1-phosphate (GlcN-1-P) by glucosamine (GlcN) phosphate mutase; iii) acetylation of GlcN-1-P by GlcN-1-P acetyltransferase to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P); and iv) synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP by GlcNAc-1-P uridyltransferase (also called UDP-GlcNAc pyrophosphorylase) (6Dobrogosz W.J. J. Bacteriol. 1968; 95: 578-584Crossref PubMed Google Scholar, 7White R.J. Biochem. J. 1968; 106: 847-858Crossref PubMed Scopus (116) Google Scholar, 8Freese E.B. Cole R.M. Klofat W. Freese E. J. Bacteriol. 1970; 101: 1046-1062Crossref PubMed Google Scholar). The Escherichia coli GlmS gene encodes glutamine:Fru-6-P amidotransferase (9Wu H.C. Wu T.C. J. Bacteriol. 1971; 105: 455-466Crossref PubMed Google Scholar, 10Walker J.E. Gay N.J. Saraste M. Eberle A.N. Biochem. J. 1984; 224: 799-815Crossref PubMed Scopus (107) Google Scholar, 11Dutka-Malen S. Mazodier P. Badet B. Biochimie (Paris). 1988; 70: 287-290Crossref PubMed Scopus (51) Google Scholar). E. coli GlmU specifies a bifunctional protein with GlcN-1-P acetyltransferase and UDP-GlcNAc pyrophosphorylase activities (12Mengin-Lecreulx D. Heijenoort J. J. Bacteriol. 1993; 175: 6150-6157Crossref PubMed Google Scholar, 13Mengin-Lecreulx D. Heijenoort J. J. Bacteriol. 1994; 176: 5788-5795Crossref PubMed Scopus (166) Google Scholar). In yeast Saccharomyces cerevisiae, Fru-6-P is converted either into GlcN-6-P by glutamine:Fru-6-P amidotransferase or into mannose-6-phosphate by phosphomannose isomerase. GFA1 andPMI have been shown to be the genes for glutamine:Fru-6-P amidotransferase and phosphomannose isomerase, respectively (14Watzele G. Tanner W. J. Biol. Chem. 1989; 264: 8753-8758Abstract Full Text PDF PubMed Google Scholar, 15Smith D.J. Proudfoot A. Friedli L. Klig L.S. Paravicini G. Payton M.A. Mol. Cell. Biol. 1992; 12: 2924-2930Crossref PubMed Scopus (58) Google Scholar). Then, GlcN-6-P is N-acetylated by an acetylase to become GlcNAc-6-P, which is further converted into GlcNAc-1-P by GlcNAc phosphate mutase (16Cabib E. Tanner W. Loewus F.A. Encyclopedia of Plant Physiology, Carbohydorate II: Extracellular carbohydorates. 13B. Springer-Verlag, Heidelberg1981: 395-416Google Scholar). S. cerevisiae harbors four different hexosephosphate mutase genes, PGM1 (17Oh D. Hopper J.E. Mol. Cell. Biol. 1990; 10: 1415-1422Crossref PubMed Scopus (48) Google Scholar), PGM2(18Boles E. Liebetrau W. Hofmann M. Zimmermann F.K. Eur. J. Biochem. 1994; 220: 83-96Crossref PubMed Scopus (82) Google Scholar), SEC53 (19Kepes F. Schekman R. J. Biol. Chem. 1988; 263: 9155-9161Abstract Full Text PDF PubMed Google Scholar), and AGM1 (20Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). Among them,AGM1 is responsible for the interconversion of GlcNAc-6-P and GlcNAc-1-P (20Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). Interestingly, Agm1p has dual substrate specificity; it also converts glucose-6-phosphate to glucose-1-phosphate (Glc-1-P) (20Hofmann M. Boles E. Zimmermann F.K. Eur. J. Biochem. 1994; 221: 741-747Crossref PubMed Scopus (62) Google Scholar). Finally, UDP-GlcNAc is produced from GlcNAc-1-P by UDP-GlcNAc pyrophosphorylase. However, the eukaryotic genes for GlcN-6-P acetylase and UDP-GlcNAc pyrophosphorylase remain unidentified. On the other hand, there are three UDP-sugar pyrophosphorylase genes inS. cerevisiae reported to date. GAL7 (21Tajima M. Nogi Y. Fukasawa T. Yeast. 1985; 1: 67-77Crossref PubMed Scopus (147) Google Scholar) andUGP1 (22Daran J.M. Dallies N. Thines-Sempoux D. Paquet V. Francois J. Eur. J. Biochem. 1995; 233: 520-530Crossref PubMed Scopus (107) Google Scholar) encode UDP-galactose (UDP-Gal) pyrophosphorylase and UDP-glucose (UDP-Glc) pyrophosphorylase, respectively. Recently, VIG9 was identified as the GDP-mannose (GDP-Man) pyrophosphorylase gene by functional complementation using the glycosylation defective vig9-1mutant (23Hashimoto H. Sakakibara A. Yamasaki M. Yoda K. J. Biol. Chem. 1997; 272: 16308-16314Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and the possible amino acid sequence motif for the active site of UDP-sugar pyrophosphorylase is proposed. Because all of these enzymes preserve substrate specificity to a certain type of sugar, there should be an enzyme specific to GlcNAc-1-P. In an attempt to identify the gene for UDP-GlcNAc pyrophosphorylase, we searched the S. cerevisiae genome data base and found that the protein specified by YDL103C. The Candida albicans and human homologs were also isolated and characterized. From sequence comparison and mutation analysis, the probable catalytic residues of UDP-sugar pyrophosphorylases are proposed. An amino acid sequence motif of LXXGXGTXMXXXXPK whereX represents any amino acid was obtained by comparing the amino acid sequences of E. coli GlmUp (EcGlm1p), S. cerevisiae Ugp1p (ScUgp1p), and Homo sapiens Ugp1p (HsUgp1p), and was used to search the S. cerevisiae genome data bases. The entire open reading frame of ScUAP1(originally designated YDL103C) was amplified by polymerase chain reaction using the S. cerevisiae genomic DNA extracted from strain A451 (MATα can1, aro7, can1, leu2, trp1, ura3) as a template, and cloned at the XbaI site of pUC18 or pYEUra3 (Toyobo) generating pUC-ScUAP1 and pYEU-ScUAP1, respectively. Primers used for polymerase chain reaction were 5′-AGATCTAGAATGACTGACACAAAACAGCT-3′ and 5′-AGATCTAGATTATTTTTCTAATACTATAC-3′. The C. albicans and human homologs of ScUAP1 were cloned by screening a C. albicans genomic DNA library and a human testis cDNA library using the 1.4-kilobaseEcoRI-EcoRI fragment of ScUAP1 as a probe. Hybridization and washing of the filters were carried out under stringent conditions (20 mm sodium phosphate (pH 7.2), 5× SSC (1× SSC contains 150 mm NaCl and 15 mmsodium 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). Bacterial cells and phages that were strongly hybridized with the probe DNA were collected. After the third screening, DNA was extracted from bacterial cells and phages, and the insert DNA was cloned at the SmaI site of pUC19 for further plasmid construction. Radiolabeling of the probe DNA was performed by the random priming method using α-32PdCTP (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and DNA sequencing was carried out as described elsewhere (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Construction of the C. albicans genomic DNA library was already reported (25Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar). A human testis cDNA library was purchased fromCLONTECH (USA). The coding regions of ScUAP1, CaUAP1,HsUAP1, and ScAGM1 were cloned at theEcoRI (for ScUAP1 and ScAGM1) orSmaI (for CaUAP1 and HsUAP1) site of pGEX2T (26Smith 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 let them express recombinant yeast and human proteins as a fusion product with glutathione S-transferase (GST). Induction and expression of the recombinant Uap1 proteins was carried out with isopropyl β-d-thio-galactopyranoside as described (25Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar, 26Smith 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-thio-galactopyranoside, the bacterial cells were harvested, suspended in a buffer containing 20 mmTris-HCl (pH 7.5), 0.5 mm EDTA, 50 mm NaCl, 10 mm β-mercaptoethanol, 10%(v/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, and lysed by sonication. After cell debris were removed by centrifugation at 15,000 ×g at 4 °C for 30 min, GST-Uap1 and GST-Agm1 fusion proteins were purified by glutathione Sepharose CL-4B column chromatography, as described (26Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The primers used for amplifying the ScAGM1 open reading frame (ORF) were 5′-CGGGAATTCATAAGGTTGATTACGAGCAAT-3′ and 5′-ATTGAATTCTCAAGCAGATGCCTTAACGTG-3′. An assay for UDP- GlcNAc pyrophosphorylase was performed in a 20 μl standard reaction mixture containing 50 mm Tris-HCl (pH 8.3), 5 mm MgCl2, 20 μm GlcNAc-1-P, 10% (v/v) glycerol, and 0.1 μm α-32PUTP (specific activity 1 × 103 cpm/pmol) and 0.1 of the recombinant proteins at 30 °C for 10 μl of reaction mixture were and were by thin layer in a that was by of of and of in of K. E. Biochem. PubMed Scopus Google Scholar). The were by An assay was carried out in μl of reaction mixture containing 50 mm Tris-HCl (pH 8.3), 5 mm MgCl2, μm 20 μm GlcNAc-1-P, 10% (v/v) glycerol, 1 and 0.1 of the recombinant enzyme. After at 30 °C for 10 min, μl of the containing and (v/v) in was to the reaction which was by at for 5 phosphate from the and the enzyme activity was by at The entire was cloned at the XbaI site of pUC18 and pYEUra3 of the generating pUC-ScUAP1 and pYEU-ScUAP1, respectively. the of the ScUAP1 in pUC-ScUAP1 was and by the generating The strain was with pYEU-ScUAP1, and were further transfected with that been with The resulting which in in were and used strain trp1, leu2, The S. cerevisiae null strain was obtained by a to that for the ScUAP1 The entire of ScAGM1 was cloned at the XbaI site of pUC18 and generating and respectively. The of in was by generating cells were with and with that been previously with The resulting which in in were and used as strain leu2, the of and the to ScUAP1, the entire and the ScUAP1 were cloned in a fragment the ScUAP1 was at the site of the of CaUAP1 and from plasmid was under the control of the ScUAP1 The resulting plasmids were transfected into cells. After of cells in the of were to containing and further for A of the an alanine substitution for Gly112, Gly114, Thr115, Arg116, Pro122, or Lys123 were by the dual method as described T. T. Y. M. Gene (Amst.). 1995; PubMed Scopus Google Scholar) with The entire of the ScUAP1 gene was cloned at the site of using and hybridized with containing the The resulting ScUAP1 genes were from the and at the site of and the site of All the were by sequencing the UDP-sugar pyrophosphorylase activities are in In S. cerevisiae, the GAL7 (21Tajima M. Nogi Y. Fukasawa T. Yeast. 1985; 1: 67-77Crossref PubMed Scopus (147) Google Scholar), J.M. Dallies N. Thines-Sempoux D. Paquet V. Francois J. Eur. J. Biochem. 1995; 233: 520-530Crossref PubMed Scopus (107) Google Scholar), and VIG9 (23Hashimoto H. Sakakibara A. Yamasaki M. Yoda K. J. Biol. Chem. 1997; 272: 16308-16314Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) genes have been shown to encode pyrophosphorylase, pyrophosphorylase, and pyrophosphorylase, the gene for UDP-GlcNAc to be of the amino acid sequences E. coli UDP-GlcNAc pyrophosphorylase and S. cerevisiae pyrophosphorylase identified an amino acid sequence represents any amino In an attempt to identify the S. cerevisiae UDP-GlcNAc pyrophosphorylase we searched the yeast data base and found that encode proteins with a sequence to the amino acid motif is identical to which has been shown to be the pyrophosphorylase we specifies UDP-GlcNAc pyrophosphorylase. The protein was expressed in E. coli as a fusion protein with and purified by column using The purified fusion protein produced when with GlcNAc-1-P and The is a gene for UDP-GlcNAc pyrophosphorylase, the gene was designated ScUAP1 S. cerevisiae UDP-GlcNAc pyrophosphorylase and enzyme activities of the yeast and human UDP-GlcNAc The yeast and human UDP-GlcNAc pyrophosphorylases were expressed in E. coli as a fusion with and purified with 1 of the purified recombinant proteins were a 10% gel and with The of the protein are in 0.1 of the purified recombinant proteins were with α-32PUTP and GlcNAc-1-P. The reaction were by and by The of the UDP-GlcNAc, and UTP that were under are Because to GlcNAc-1-P from we a assay by to the reaction which to the enzyme activity from the of produced after the of in the reaction it was that the fusion protein converted GlcNAc-1-P to UDP-GlcNAc in a UTP was essential for the of UDP-GlcNAc by of and were used as the substrate Because UDP-GlcNAc is an essential metabolite as a precursor of cell wall protein and in yeast (4Cabib E. Roberts R. Bowers B. Annu. Rev. Biochem. 1982; 51: 763-793Crossref PubMed Scopus (270) Google Scholar, A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (440) Google Scholar), ScUAP1 be an essential gene for it is the UDP-GlcNAc pyrophosphorylase gene in S. The S. cerevisiae null strain in which the UAP1 gene was of UAP1 whose was under the control of were The cells of S. cerevisiae null an of the yeast cells and were which is a to that by a null mutation the gene for GlcNAc phosphate mutase is that is a UDP-GlcNAc pyrophosphorylase gene inS. cerevisiae and that the resulting from of the functional UAP1 in the cell into the of UDP-GlcNAc pyrophosphorylase, we to homologs from the C. albicans as as from screening a C. albicans genomic DNA library and a human testis cDNA library with ScUAP1 DNA as a C. albicans and the human homologs of ScUAP1, were cloned and The of ScUAP1, and highly to other Interestingly, the cDNA was identical to the previously cDNA whose product is as an E. Biol. 1994; PubMed Scopus Google Scholar). of the recombinant and which were expressed in E. coli as a fusion with GST, possessed UDP-GlcNAc pyrophosphorylase activities that CaUAP1 and indeed specify UDP-GlcNAc pyrophosphorylase. expression or under the control of the of S. cerevisiae cells in the of it that the C. albicans and human UAP1 the substrate specificity of UDP-GlcNAc pyrophosphorylase using ScUap1p converted GlcNAc-1-P into UDP-GlcNAc in the of UTP GlcNAc-6-P, or as a substrate 5 the enzyme a whose to that of from Glc-1-P, the dual substrate of However, was as shown in B. ScUAP1 It is that the interconversion of GlcNAc-6-P and GlcNAc-1-P precedes the in to the yeast GlcNAc phosphate mutase to the reaction shown in together with ScUap1p produced UDP-GlcNAc from GlcNAc-6-P, It is also that hexosephosphate either as an or a for the catalytic reaction Biochem. 1985; PubMed Scopus Google Scholar, C. C. W. 1997; PubMed Scopus Google Scholar). we the of the synthesis of UDP-GlcNAc from The analysis of the that was essential for the interconversion of GlcNAc-6-P and GlcNAc-1-P, UDP-GlcNAc was produced from GlcNAc-6-P by Agm1p and Uap1p in the of However, further of the of for the reaction by revealed that the by of the mutase reaction was when the was of the amino acid sequences UDP-sugar pyrophosphorylases revealed that the amino acid and of ScUap1p sequence with other UDP-sugar pyrophosphorylases the of for the catalytic activity, the highly conserved amino in Gly112, Gly114, Thr115, Arg116, Pro122, and Lys123 were by was for the type ScUap1p, all the enzymes were expressed as a fusion with and purified by column Gly114, Thr115, and are also highly conserved in known UDP-sugar of these amino by alanine the enzyme In substitution of alanine for Gly112, Arg116, or Lys123 severely diminished the activity other a to GlcNAc-1-P and all of and to the S. cerevisiae null of the in to UTP it was that serves as a site for GlcNAc-1-P, and that Gly112, Arg116, and Lys123 are possible catalytic ScUap1p activity of alanine substitution for the conserved amino The proteins an alanine substitution for of the amino that are highly conserved in UDP-sugar pyrophosphorylases were expressed as a fusion with and purified with 1 of the type and the proteins were a 10% gel and with The of is by the 0.1 of the purified and the proteins were with GlcNAc-1-P and and the of the phosphate that the enzyme activities were with and of the and of the type and enzymes to GlcNAc-1-P were from the of from The to UTP were also in the in a The and of the type and enzymes to GlcNAc-1-P were from the of from The to UTP were also in the In we have identified the eukaryotic UDP-GlcNAc pyrophosphorylase The amino acid sequences of the yeast and human enzymes are and C. human enzymes S. cerevisiae the yeast enzyme to Glc-1-P, ScUap1p a substrate specificity to GlcNAc-1-P, the enzyme GlcNAc-1-P. In of ScUAP1 the by a of S. the enzyme GlcNAc-6-P, together with it produced UDP-GlcNAc from GlcNAc-6-P, that the GlcNAc phosphate mutase reaction precedes in UDP-GlcNAc However, we out the that the results of the and the be the in the assay the reaction was by UDP-GlcNAc pyrophosphorylase and GlcN-1-P acetyltransferase activities are in E. coli GlmUp (9Wu H.C. Wu T.C. J. Bacteriol. 1971; 105: 455-466Crossref PubMed Google Scholar). It has also been that the is responsible for the and acetylase activity in the of GlmUp D. Heijenoort J. J. Bacteriol. 1994; 176: 5788-5795Crossref PubMed Scopus (166) Google Scholar). bacterial UDP-GlcNAc pyrophosphorylase, the eukaryotic enzymes to be ScUap1p GlcN-1-P as the substrate and the of GlmUp sequence to any UDP-GlcNAc pyrophosphorylase. it is that in GlcN-6-P is by an as enzyme and the mutase reaction GlcNAc-1-P. and a as a which serves as a phosphate to the enzyme by Biochem. 1985; PubMed Scopus Google Scholar, C. C. W. 1997; PubMed Scopus Google Scholar). In was to produce GlcNAc-1-P in the of a of was possible for is that a of the recombinant was already and However, is with the by C. C. W. 1997; PubMed Scopus Google Scholar) that the and yeast enzymes a as a Sequence of the UDP-sugar revealed that there is a the amino acid sequence is highly conserved of the known UDP-sugar substitution for Gly112, Arg116, or diminished the enzyme activity and to the type ScUAP1 strongly suggesting that these amino are catalytic Among these three amino acids, was shown to be a possible site to GlcNAc-1-P, an In human pyrophosphorylase, it was that a mutation of to the enzyme activity and cellular M. A. B. P. M. C. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Sequence comparison of the and proteins that the human Ugp1p to of the yeast it is that of the human Ugp1p also serves as a which is known as pyrophosphorylase, sequence to known UDP-sugar and the conserved amino essential for the catalytic activity of ScUap1p are found in (21Tajima M. Nogi Y. Fukasawa T. Yeast. 1985; 1: 67-77Crossref PubMed Scopus (147) Google Scholar). that the catalytic of from of other UDP-sugar The human UDP-GlcNAc pyrophosphorylase cDNA to be identical to the the to be it encodes an expressed in and is in human E. Biol. 1994; PubMed Scopus Google Scholar). is expressed in and of were in and E. Biol. 1994; PubMed Scopus Google Scholar). The testis a of and UDP-GlcNAc pyrophosphorylase human further In there is an which from by a in the The of was in of were in and E. Biol. 1994; PubMed Scopus Google Scholar). it also be of to the the UDP-GlcNAc pyrophosphorylase K. for data base search and sequence Y. and Y. for with the and S. for reading the