The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases
GENE CLONING, PROTEIN EXPRESSION, AND CATALYTIC MECHANISM*

Toshiyuki Mio, Tomio Yabe, Mikio Arisawa, and Hisafumi Yamada-OkabeDagger

From the Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A search of the yeast data base for a protein homologous to Escherichia coli UDP-N-acetylglucosamine pyrophosphorylase yielded UAP1 (UDP-N-acetylglucosamine pyrophosphorylase), 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, both C. albicans and Homo sapiens UAP1 (CaUAP1 and HsUAP1) rescued the ScUAP1-deficient S. cerevisiae cells. All the recombinant ScUap1p, CaUap1p, and HsUap1p possessed UDP-N-acetylglucosamine pyrophosphorylase activities in vitro. The yeast Uap1p utilized N-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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

UDP-N-acetylglucosamine (UDP-GlcNAc1) 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 (1-3). In eukaryotes, it serves as the substrate of chitin synthase, whose product is shown to be essential for fungal cell wall (4). It is also used in the GlcNAc moiety of N-linked glycosylation and the GPI-anchor of cellular proteins (5).

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) (6-8). The Escherichia coli GlmS gene encodes glutamine:Fru-6-P amidotransferase (9-11). E. coli GlmU specifies a bifunctional protein with GlcN-1-P acetyltransferase and UDP-GlcNAc pyrophosphorylase activities (12, 13).

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 and PMI have been shown to be the genes for glutamine:Fru-6-P amidotransferase and phosphomannose isomerase, respectively (14, 15). 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 (16). S. cerevisiae harbors four different hexosephosphate mutase genes, PGM1 (17), PGM2 (18), SEC53 (19), and AGM1 (20). Among them, AGM1 is responsible for the interconversion of GlcNAc-6-P and GlcNAc-1-P (20). Interestingly, Agm1p has dual substrate specificity; it also converts glucose-6-phosphate to glucose-1-phosphate (Glc-1-P) (20). 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 in S. cerevisiae reported to date. GAL7 (21) and UGP1 (22) 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 vig9-1 mutant (23), 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Data Base Search and Screening of DNA Libraries-- An amino acid sequence motif of LXXGXGTXMXXXXPK where X 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 (MATalpha 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-kilobase EcoRI-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 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). 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 [alpha -32P]dCTP (24), and DNA sequencing was carried out as described elsewhere (24). Construction of the C. albicans genomic DNA library was already reported (25). A human testis cDNA library was purchased from CLONTECH (USA).

Expression and Purification of the Recombinant Proteins-- The coding regions of ScUAP1, CaUAP1, HsUAP1, and ScAGM1 were cloned at the EcoRI (for ScUAP1 and ScAGM1) or SmaI (for CaUAP1 and HsUAP1) site of pGEX2T (26), 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 beta -D-thio-galactopyranoside as described (25, 26). At 4 h after the addition of isopropyl-beta -D-thio-galactopyranoside, the bacterial cells were harvested, suspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 50 mM NaCl, 10 mM beta -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 (26) 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'.

Assays for UDP-GlcNAc Pyrophosphorylase-- 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 [alpha -32P]UTP (specific activity 1 × 103 cpm/pmol) and approximately 0.1 µg of the indicated recombinant proteins at 30 °C for 10 min. 2 µl of each reaction mixture were spotted onto polyethyleneimine cellulose plates, and nucleotide sugars were separated by thin layer chromatography (TLC) in a solution that was prepared by mixing 6 g of Na2B4O7/10H2O, 3 g of H3BO3, and 25 ml of ethylene glycol in 70 ml of H2O (27). The radioactive spots were visualized by autoradiography. An alternative high flux assay was carried out in 90 µl of reaction mixture containing 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 25 µM UTP, 20 µM GlcNAc-1-P, 10% (v/v) glycerol, 1 mM dithiothreitol, 0.4 units/ml pyrophosphatase (Sigma), and approximately 0.1 µg of the recombinant enzyme. After incubation at 30 °C for 10 min, 100 µl of the color reagent containing 0.03% (w/v) malachite green, 0.2% (w/v) ammonium molybdate, and 0.05% (v/v) Triton X-100 in 0.7 N HCl was added to the reaction mixture, which was followed by incubation at room temperature for 5 min. Inorganic phosphate derived from the pyrophosphate and thereby representing the enzyme activity was quantified by measuring optimal density at 655 nm.

Yeast Strains and Plasmids-- The entire ORF of ScUAP1 was cloned at the XbaI site of pUC18 and pYEUra3 (downstream of the GAL1 promoter), generating pUC-ScUAP1 and pYEU-ScUAP1, respectively. Then the 539-base pair StuI-BalI region of the ScUAP1 ORF in pUC-ScUAP1 was excised and replaced by the LEU2 gene, generating pUC-ScUAP1L. The haploid strain YPH499 (MATa ura3, lys2, ade2, trp1, his3, leu2) was transformed with pYEU-ScUAP1, and ura+ transformants were further transfected with pUC-ScUAP1L that had been digested with XbaI. The resulting ura+ leu+ transformants, which grew in galactose medium but not in glucose medium, were collected and used as uap1Delta strain (MATa ura3, lys2, ade2, trp1, his3, leu2, uap1Delta ::LEU2 UAP1-URA3).

The S. cerevisiae agm1Delta null mutant strain was obtained by a means similar to that for the ScUAP1 depletion. The entire ORF of ScAGM1 was cloned at the XbaI site of pUC18 and pYEUra3, generating pUC-ScAGM1 and pYEU-ScAGM1, respectively. The 1.4-kilobase BalI-BglII region of the ScAGM1 ORF in pUC-ScAGM1 was replaced by LEU2, generating pUC-AGM1L. YPH499 cells were transformed with pYEU-ScAGM1 and then with pUC-ScAGM1L that had been previously digested with XbaI. The resulting ura+ leu+ transformants, which grew in galactose medium but died in glucose medium, were collected and used as agm1Delta strain (MATa ura3, lys2, ade2, trp1, his3, leu2, agm1Delta ::LEU2 AGM1-URA3).

To test the ability of CaUAP1, HsUAP1, and the mutant ScUAP1 to complement ScUAP1, the entire ORFs of CaUAP1, HsUAP1, and the mutant ScUAP1 were cloned in pRS414-1 where a 2.0-kilobase BglII-XbaI fragment encompassing the ScUAP1 promoter was inserted at the BamHI site of pRS414 (Stratagene). Thus, the transcription of CaUAP1 and HsUAP1 from this plasmid was under the control of the ScUAP1 promoter. The resulting plasmids were transfected into uap1Delta cells. After selection of trp+ cells in the presence of galactose, they were transferred to plates containing glucose and further cultured for 3 days.

Site-directed Mutagenesis-- A series of the ScUAP1 mutants harboring an alanine substitution for Gly111, Gly112, Gly114, Thr115, Arg116, Leu117, Pro122, or Lys123 were generated by the oligonucleotide-directed dual amber method as described (28) with Mutan-Express KmTM (Takara). The entire ORF of the ScUAP1 gene was cloned at the EcoRI site of pKF18k (Takara) using EcoRI linker and hybridized with oligonucleotides containing the indicated mutations. The resulting mutant ScUAP1 genes were excised from the vector and ligated at the EcoRI site of pGEX-2T and the BamHI site of pRS414-1. All the mutations were confirmed by sequencing the DNA.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of the Yeast UDP-GlcNAc Pyrophosphorylase Gene-- Three distinct UDP-sugar pyrophosphorylase activities are present in yeast. In S. cerevisiae, the GAL7 (21), UGP1 (22), and VIG9 (23) genes have been shown to encode UDP-Gal pyrophosphorylase, UDP-Glc pyrophosphorylase, and GDP-Man pyrophosphorylase, respectively, but the gene for UDP-GlcNAc remains to be established. Comparison of the amino acid sequences between E. coli UDP-GlcNAc pyrophosphorylase (GlmUp) and S. cerevisiae UDP-Glc pyrophosphorylase (Ugp1p) identified an amino acid sequence motif, L(X)2GXGTXM(X)4PK, where X represents any amino acid. In an attempt to identify the S. cerevisiae UDP-GlcNAc pyrophosphorylase gene, we searched the yeast data base and found that PSA1 and YDL103C could encode proteins with a sequence similar to the above amino acid motif (Fig. 1). PSA1 is identical to VIG9, which has been shown to be the GDP-Man pyrophosphorylase gene. Accordingly, we asked whether YDL103C specifies UDP-GlcNAc pyrophosphorylase. The Ydl103c protein was expressed in E. coli as a fusion protein with GST and purified by affinity column chromatography using glutathione-Sepharose CL-4B. The purified GST-Ydl103c fusion protein produced [32P]UDP-GlcNAc when incubated with GlcNAc-1-P and [alpha -32P]UTP, whereas GST alone did not (Fig. 2). The above result demonstrates that YDL103C is a gene for UDP-GlcNAc pyrophosphorylase, and, therefore, the gene was designated ScUAP1 (the S. cerevisiae UDP-GlcNAc pyrophosphorylase gene 1).


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Fig. 1.   Predicted amino acid sequences of the yeast and human UDP-GlcNAc pyrophosphorylases. Amino acid sequences of UDP-GlcNAc pyrophosphorylases of S. cerevisiae (ScUap1p or Ydl103cp), C. albicans (CaUap1p), and H. sapiens (HsUap1p) are aligned using the FASTA and BLAST programs. Identical amino acids among the three proteins are boxed. The amino acid sequence that matched L(X)2GXGTXM(X)4PK motif is underlined.


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Fig. 2.   Expression and enzyme activities of the yeast and human UDP-GlcNAc pyrophosphorylases. The yeast and human UDP-GlcNAc pyrophosphorylases were expressed in E. coli as a fusion with GST and purified with glutathione-Sepharose beads. A, approximately 1 µg of the purified recombinant proteins were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, GST; lane 2, GST-ScUap1p; lane 3, GST-CaUap1p; lane 4, GST-HsUap1p; lane 5, GST-ScAgm1p. The positions of the protein size markers are indicated in kDa. B, approximately 0.1 µg of the purified recombinant proteins were incubated with [alpha -32P]UTP and GlcNAc-1-P. The reaction products were separated by polyethyleneimine cellulose TLC and visualized by autoradiography. The positions of the UDP-GlcNAc, UDP-Glc, and UTP that were visualized under UV light are indicated. Lane 1, no protein; lane 2, GST; lane 3, GST-ScUap1p; lane 4, GST-CaUap1p; lane 5, GST-HsUap1p.

Because uridyltransfer to GlcNAc-1-P releases pyrophosphate from UTP, we developed a conventional high-flux assay by adding pyrophosphatase to the reaction mixture, which allows us to estimate the enzyme activity from the amounts of inorganic phosphates produced after the hydrolysis of pyrophosphates in the reaction mixture. By this assay, it was demonstrated that the GST-ScUap1 fusion protein converted GlcNAc-1-P to UDP-GlcNAc in a dose-dependent manner (see below). Moreover, UTP was essential for the production of UDP-GlcNAc by ScUap1p; none of ATP, GTP, and CTP were used as the substrate (not shown).

Because UDP-GlcNAc is an essential metabolite serving as a precursor of cell wall chitin, protein N-glycosylation, and GPI anchor in yeast (4, 5), ScUAP1 may be an essential gene for viability if it is the only UDP-GlcNAc pyrophosphorylase gene in S. cerevisiae. The S. cerevisiae uap1Delta null mutant strain in which the endogenous UAP1 gene was disrupted, but where episomal copies of UAP1 whose transcription was under the control of GAL1 promoter were maintained, grew on galactose plates but died on glucose plates. The cells of S. cerevisiae uap1Delta null mutant displayed an aberrant morphology; most of the yeast cells fully swelled and some were lysed, which is a phenotype quite similar to that caused by a null mutation of AGM1, the gene for GlcNAc phosphate mutase (Fig. 3). This is suggestive that the ScUAP1 is a sole UDP-GlcNAc pyrophosphorylase gene in S. cerevisiae and that the most apparent defect resulting from depletion of the functional UAP1 occurred in the cell wall.


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Fig. 3.   Aberrant morphology caused by the depletion of ScUAP. S. cerevisiae cells of the wild type, uap1Delta harboring pYEU-ScUAP1, and agm1Delta harboring pYEU-ScAGM1 were spread on agar plates containing galactose or glucose and incubated at 30 °C. Photographs were taken at 24 h.

Identification of the UDP-GlcNAc Pyrophosphorylase Genes of Other Organisms-- To gain more insight into the characteristics of UDP-GlcNAc pyrophosphorylase, we intended to isolate the ScUAP1 homologs from the pathogenic fungus C. albicans as well as from human. By screening a C. albicans genomic DNA library and a human testis cDNA library with ScUAP1 DNA as a probe, CaUAP1, and HsUAP1, C. albicans (Ca) and the human (Hs) homologs of ScUAP1, were cloned and sequenced. The predicted products of ScUAP1, CaUAP1, and HsUAP1 are highly related to each other (Fig. 1). Interestingly, the cloned HsUAP1 cDNA was identical to the previously reported AGX1 cDNA whose product is implicated as being an antigen causing male infertility (29). Both of the recombinant CaUap1p and HsUap1p, which were expressed in E. coli as a fusion with GST, possessed UDP-GlcNAc pyrophosphorylase activities (Fig. 2), confirming that CaUAP1 and HsUAP1 indeed specify UDP-GlcNAc pyrophosphorylase. Furthermore, expression of CaUAP1 or HsUAP1 under the control of the ScUAP1 promoter supported the growth of the UAP1-deficient S. cerevisiae cells even in the presence of glucose. Thus, it appears that both the C. albicans and human UAP1 functionally complement ScUAP1 (Fig. 4).


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Fig. 4.   Functional complementation of ScUAP1 by the C. albicans and human UAP1. S. cerevisiae uap1Delta cells harboring pYEU-ScUAP1 were further transformed with pRS414-1 bearing ScUAP1, CaUAP1, or HsUAP1. Transfectants were spread on agar plates containing galactose or glucose and incubated at 30 °C for 3 days.

Substrate Specificity of UDP-GlcNAc Pyrophosphorylase-- We next examined the substrate specificity of UDP-GlcNAc pyrophosphorylase using ScUap1p. ScUap1p reproducibly converted GlcNAc-1-P into UDP-GlcNAc in the presence of UTP but did not utilize GlcNAc-6-P, galactose-1-phosphate (Gal-1-P) or mannose-1-phosphate (Man-1-P) as a substrate (Fig. 5A). Unexpectedly, the enzyme generated a spot whose mobility corresponded to that of UDP-Glc from Glc-1-P, indicating the dual substrate utility of Uap1p. However, Glc-1-P was much less efficient as shown in Fig. 5B. Consequently, ScUAP1 did not complement ScUGP1 (data not shown).


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Fig. 5.   Substrate specificity of ScUap1p. A, approximately 0.1 µg of the purified GST-ScUap1p was incubated with [alpha -32P]UTP and 20 µM of each sugar nucleotide. The reaction products were separated by polyethyleneimine cellulose TLC and visualized by autoradiography. The positions of the UDP-GlcNAc, UDP-Glc, and UTP that were visualized under UV light are indicated. Lane 1, none; lane 2, GlcNAc-1-P; lane 3, GlcNAc-6-P; lane 4, GlcN-1-P; lane 5, Glc-1-P; lane 6, Gal-1-P; lane 7, GalNAc-1-P; lane 8, Man-1-P. B, the indicated amounts of the purified GST-ScUap1p were incubated with 20 µM of the indicated nucleotide sugar, UTP, and pyrophosphatase. The amounts of the released inorganic phosphate that represent the enzyme activities were determined with malachite green and ammonium molybdate.

It is believed that the interconversion of GlcNAc-6-P and GlcNAc-1-P precedes the uridyltransfer in vivo. This prompted us to add the yeast GlcNAc phosphate mutase (ScAgm1p) to the reaction mixture. As shown in Fig. 6A, together with ScAgm1p, ScUap1p produced UDP-GlcNAc from GlcNAc-6-P, whereas ScAgm1p alone did not. It is also demonstrated that hexosephosphate mutases require Glc-1,6-P2 either as an activator or a cofactor for the catalytic reaction (30, 31). Therefore, we examined the effect of Glc-1,6-P2 on the synthesis of UDP-GlcNAc from GlcNAc-6-P. The TLC analysis of the products indicated that Glc-1,6-P2 was not essential for the interconversion of GlcNAc-6-P and GlcNAc-1-P, because UDP-GlcNAc was produced from GlcNAc-6-P by Agm1p and Uap1p even in the absence of Glc-1,6-P2 (Fig. 6A). However, further assessment of the importance of Glc-1,6-P2 for the reaction by ScAgm1p revealed that the enhancement by Glc-1,6-P2 of the mutase reaction was significant when the ScAgm1p concentration was rather low (Fig. 6B).


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Fig. 6.   Production of UDP-GlcNAc from GlcNAc-6-P by ScUap1p and ScAgm1p. A, approximately 0.1 µg of the purified GST-ScUap1p (ScUap1p) was incubated with [alpha -32P]UTP and 20 µM each of the indicated sugar nucleotides in the presence or absence of approximately 0.1 µg of the purified GST-ScAgm1p. The reaction products were separated by polyethyleneimine cellulose TLC and visualized by autoradiography. The positions of the UDP-GlcNAc, UDP-Glc, and UTP that were visualized under UV light are indicated. It should be noted that UDP-Glc, which appeared in the presence of ScUap1p and Glc-1,6-P2, was formed from a trace of Glc-1-P in the Glc-1,6-P2. B, the indicated amounts of the purified GST-ScAgm1p were incubated with approximately 0.1 µg of the purified GST-ScUap1p, GlcNAc-6-P, and UTP in the presence (open circle ) or absence (triangle ) of 20 mM Glc-1,6-P2. The amounts of the released inorganic phosphate that represent the enzyme activities were determined with malachite green and ammonium molybdate.

Possible Active Sites of ScUap1p-- Comparison of the amino acid sequences among UDP-sugar pyrophosphorylases revealed that the region between amino acid positions 111 and 123 of ScUap1p shares significant sequence identity with other UDP-sugar pyrophosphorylases (Fig. 7). To verify the importance of this region for the catalytic activity, the highly conserved amino acids in this region, Gly111, Gly112, Gly114, Thr115, Arg116, Leu117, Pro122, and Lys123 were replaced by alanine. As was done for the wild type ScUap1p, all the mutant enzymes were expressed as a fusion with GST and purified by affinity column chromatography (Fig. 8A). Although Gly114, Thr115, and Pro122 are also highly conserved in known UDP-sugar pyrophosphorylases, replacement of these amino acids by alanine only weakly impaired the enzyme activity. In contrast, substitution of alanine for Gly112, Arg116, or Lys123 severely diminished the activity (Fig. 8B). Furthermore, G112A but not other mutants displayed a higher Km value to GlcNAc-1-P (Table I), and all of G112A, R116A, and K123A failed to rescue the S. cerevisiae uap1Delta null mutant (data not shown). None of the mutations significantly affected the Km values in response to UTP (Table I). Taken together, it was proposed that Gly112 serves as a binding site for GlcNAc-1-P, and that Gly112, Arg116, and Lys123 are possible catalytic residues.


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Fig. 7.   Comparison of the amino acid sequences of UDP-sugar pyrophosphorylases. Amino acid sequences of ScUap1p are compared with those of other UDP-sugar pyrophosphorylases using the FASTA and BLAST programs. Identical amino acids among all the proteins listed here are indicated by bold letters. Amino acids that were replaced by alanine are marked by +. ScUap1p, S. cerevisiae UDP-GlcNAc pyrophosphorylase; CaUap1p, C. albicans UDP-GlcNAc pyrophosphorylase; HsUap1p, H. sapiens UDP-GlcNAc pyrophosphorylase; ScUgp1p, S. cerevisiae UDP-Glc pyrophosphorylase (22); EcGlmUp, E. coli UDP-GlcNAc pyrophosphorylase (12, 13); HsUgp1p, H. sapiens UDP-Glc pyrophosphorylase (33); EcUgp1p, E. coli UDP-Glc pyrophosphorylase (34); ScVig9p, S. cerevisiae GDP-Man pyrophosphorylase (23).


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Fig. 8.   Effects on ScUap1p activity of alanine substitution for the conserved amino acids. The ScUap1 mutant proteins harboring an alanine substitution for each of the amino acids that are highly conserved in UDP-sugar pyrophosphorylases were expressed as a fusion with GST and purified with glutathione-Sepharose beads. A, approximately 1 µg of the wild type and the indicated mutant proteins were separated on a 10% SDS-polyacrylamide gel (PAGE) and stained with Coomassie Brilliant Blue. The position of GST-ScUap1p is indicated by the arrowhead. B, approximately 0.1 µg of the purified GST and the indicated mutant proteins were incubated with GlcNAc-1-P and UTP, and the amounts of the released inorganic phosphate that represent the enzyme activities were determined with malachite green and ammonium molybdate.

                              
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Table I
Characteristics of the mutant ScUap1p
The Km and kcat values of the wild type and mutant enzymes to GlcNAc-1-P were determined from the amounts of pyrophosphate released from UTP. The Km values to UTP were also indicated in the right column.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this paper, we have identified the eukaryotic UDP-GlcNAc pyrophosphorylase genes. The expected amino acid sequences of the yeast and human enzymes are well conserved, and both C. albicans and human enzymes functionally complement S. cerevisiae UAP1. Although the yeast enzyme catalyzed uridyltransfer to Glc-1-P, ScUap1p displayed a reasonable substrate specificity to GlcNAc-1-P, because the enzyme utilized Glc-1-P much less efficiently than GlcNAc-1-P. In fact, overexpression of ScUAP1 did not overcome the lethal phenotype caused by a depletion of UGP1 in S. cerevisiae. Moreover, the enzyme did not recognize GlcNAc-6-P, but together with ScAgm1p, it produced UDP-GlcNAc from GlcNAc-6-P, demonstrating that the GlcNAc phosphate mutase reaction precedes uridyltransfer in UDP-GlcNAc biosynthesis. However, we cannot rule out the possibility that the results of the TLC assays and the high flux assays may not be exactly the same, because in the high flux assay the reverse reaction was eliminated by pyrophosphatase.

Both UDP-GlcNAc pyrophosphorylase and GlcN-1-P acetyltransferase activities are authentic in E. coli GlmUp (9). It has also been demonstrated that the N-terminal region is responsible for the uridyltransfer, and acetylase activity resides in the C-terminal half of GlmUp (13). Unlike bacterial UDP-GlcNAc pyrophosphorylase, the eukaryotic enzymes seem not to be bifunctional, because ScUap1p did not utilize GlcN-1-P as the substrate and the C-terminal portion of GlmUp showed no significant sequence homology to any UDP-GlcNAc pyrophosphorylase. Thus, it is likely that in yeast, GlcN-6-P is first acetylated by an as yet unidentified enzyme and then the mutase reaction generates GlcNAc-1-P.

Phosphomannomutase and phosphoglucomutase require a sugar biphosphate as a cofactor, which serves as a phosphate donor necessary to activate the enzyme by phosphorylation (30, 31). In this study, ScAgm1p was able to produce GlcNAc-1-P even in the absence of cofactor, Glc1,6-P2, if a sufficient amount of ScAgm1p was present. One possible explanation for this discrepancy is that a small portion of the recombinant ScAgm1p was already phosphorylated and thereby activated. However, this hypothesis is inconsistent with the recent report by Oesterhelt et al. (31) that the plant and yeast enzymes utilize a sugar diphosphate as a co-substrate.

Sequence comparisons of the UDP-sugar transferases revealed that there is a region where the amino acid sequence is highly conserved among most of the known UDP-sugar pyrophosphorylases. Alanine substitution for Gly112, Arg116, or Lys123 severely diminished the enzyme activity and ability to complement the wild type ScUAP1 gene, strongly suggesting that these amino acids are catalytic residues. Among these three amino acids, Gly112 was shown to be a possible binding site to GlcNAc-1-P, because G112A displayed an increased Km value. In human UDP-Glc pyrophosphorylase, it was demonstrated that a single mutation of Gly115 to Asp drastically impaired the enzyme activity and caused cellular UDP-Glc deficiency (32). Sequence comparison of the Uap and Ugp proteins reveals that Gly115 of the human Ugp1p corresponds to Gly112 of the yeast Ugp1p. Thus, it is likely that Gly115 of the human Ugp1p also serves as a Glc-1-P binding site. Curiously, Gal7p, which is known as UDP-Gal pyrophosphorylase, shares no significant sequence homology to known UDP-sugar pyrophosphorylases, and the conserved amino acids essential for the catalytic activity of ScUap1p are not found in Gal7p (21). This may imply that the catalytic mechanism of Gal7p differs from those of other UDP-sugar pyrophosphorylases.

The human UDP-GlcNAc pyrophosphorylase cDNA turned to be identical to the AGX1 cDNA. Although the physiological function of AGX1 remains to be established, it encodes an unknown antigen expressed in infertile males and is implicated in antibody-mediated human infertility (29). AGX1 is abundantly expressed in testes, and only low levels of AGX1 mRNA were detected in placenta, muscle, and liver (29). The reason why testis expresses a higher level of AGX1 mRNA and how UDP-GlcNAc pyrophosphorylase causes human male infertility await further study. In addition, there is an additional AGX cDNA, termed AGX2, which differs from AGX1 by a 48-base pair insertion in the ORF. The level of AGX2 mRNA was not remarkably increased in testis; low but similar levels of AGX2 mRNA were detected in testis, placenta, muscle, and liver (29). Therefore, it may also be of interest to study how the internal 48-base pair insertion affects the UDP-GlcNAc pyrophosphorylase activity.

    ACKNOWLEDGEMENTS

We thank K. Saitoh for data base search and sequence alignment, Y. Miyazaki and Y. Kitayama for assisting with the experiments, and S. Miwa for reading the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB011272, AB011003, AB011004.

Dagger To whom correspondence should be addressed. Tel.: 81-467-47-2242; Fax: 81-467-46-5320; E-mail: hisafumi.okabe{at}roche.com.

1 The 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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