Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase

Candida albicansglucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited K m(Gln) = 1.56 mmand K m(Fru-6-P) = 1.41 mmand catalyzed GlcN-6-P formation with k cat = 1150 min−1. The isoelectric point of 4.6 ± 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330–340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mm). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with n H = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1.21 ± 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast → mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter. Candida albicansglucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited K m(Gln) = 1.56 mmand K m(Fru-6-P) = 1.41 mmand catalyzed GlcN-6-P formation with k cat = 1150 min−1. The isoelectric point of 4.6 ± 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330–340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mm). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with n H = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1.21 ± 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast → mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter. l-Glutamine:d-fructose-6-phosphate amidotransferase (hexose-isomerizing) EC 2.6.1.16, known under a trivial name of glucosamine-6-phosphate (GlcN-6-P) 1The abbreviations used are: GlcN-6-P, d-glucosamine 6-phosphate; UDP-GlcNAc, uridine 5′-diphospho-N-acetyl-d-glucosamine; BSA, bovine serum albumin; PCR, polymerase chain reaction; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; NTCB, 2-thiocyano-5-nitrobenzoic acid; IAA, iodoacetamide; NAI, N-acetylimidazole; DEP, diethylpyrocarbonate; BD, 2,3-butanedione; kb, kilobase pair; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-4-toluene sulfonate; PLP, pyridoxal 5′-phosphate; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; Y → M, yeast-to-mycelia. synthase, catalyzes the complex reaction involving ammonia transfer and sugar phosphate isomerization: l-glutamine +d-fructose 6-phosphate → d-glucosamine 6-phosphate + l-glutamate. This reaction is the first committed step of the cytoplasmic biosynthetic pathway leading to the formation of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc). The final product of this pathway is an activated precursor of numerous macromolecules containing amino sugars, including chitin and mannoproteins in fungi, peptidoglycan and lipopolysaccharides in bacteria, and glycoproteins in mammals. GlcN-6-P synthase belongs to the class II amidotransferase family but is unique among other amidotransferases due to its apparent inability to use exogenous ammonia as a nitrogen donor (1Zalkin H. Adv. Enzymol. 1993; 66: 203-309PubMed Google Scholar). The enzyme is widely distributed in nature, and its activity has been detected in almost every organism and tissue; several genes coding for GlcN-6-P synthase have been cloned and sequenced (for review see Ref. 1Zalkin H. Adv. Enzymol. 1993; 66: 203-309PubMed Google Scholar). However, only prokaryotic GlcN-6-P synthases have been purified to apparent homogeneity from Escherichia coli and from the thermophilic bacteria Thermus thermophilus (3Badet-Denisot M.-A. Fernandez-Herrero L.A. Berenguer J. Ooi T. Badet B. Arch. Biochem. Biophys. 1997; 337: 129-136Crossref PubMed Scopus (15) Google Scholar). The E. colienzyme has been crystallized (4Obmolova G. Badet-Denisot M.-A. Badet B. Teplyakov A. J. Mol. Biol. 1994; 242: 703-705Crossref PubMed Scopus (24) Google Scholar), and a structure of the glutamine-binding domain has been elucidated (5Isupov M.N. Obmolova G. Butterworth S. Badet-Denisot M.-A. Badet B. Polikarpov I. Littlechild J.A. Teplyakov A. Structure. 1996; 4: 801-810Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Availability of the pure protein has facilitated extensive studies on its structure and molecular mechanism of the enzymatic reaction (6Badet-Denisot M.-A. Rene L. Badet B. Bull. Soc. Chim. Fr. 1993; 130: 249-255Google Scholar, 7Golinelli-Pimpaneau B. LeGoffic F. Badet B. J. Am. Chem. Soc. 1989; 111: 3029-3034Crossref Scopus (45) Google Scholar, 8Massiere F. Badet-Denisot M.-A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar). On the other hand, there is very little known of the molecular structure of eukaryotic GlcN-6-P synthases, and all the studies performed so far have been done on partially purified preparations. Several lines of evidence indicate that the eukaryotic enzyme could be different from its prokaryotic counterpart. Comparison of the available gene sequences has revealed a relatively large region (about 200 base pairs) that is lacking in the prokaryotic proteins (9Smith R.J. Milewski S. Brown A.J.P. Gooday G.W. J. Bacteriol. 1996; 178: 2320-2327Crossref PubMed Google Scholar). Eukaryotic but not prokaryotic GlcN-6-P synthases are the subject of feedback inhibition by UDP-GlcNAc (10Kornfeld R. J. Biol. Chem. 1967; 242: 3135-3141Abstract Full Text PDF PubMed Google Scholar). Sensitivity to this inhibitor inBlastocladiella emersonii (11Etchebehere L.C. Maia J.C.C. Arch. Biochem. Biophys. 1989; 272: 301-310Crossref PubMed Scopus (19) Google Scholar) and probably inAspergillus nidulans (12Borgia P.T. J. Bacteriol. 1992; 174: 384-389Crossref PubMed Google Scholar) enzymes is modulated by reversible phosphorylation/dephosphorylation mediated by protein kinase(s) and phosphatase(s). Fungal GlcN-6-P synthase is a subject of interest as a potential target in antifungal chemotherapy (13.Borowski, E., Abstracts of the 8th International Symposium on Future Trends in Chemotherapy, Tirrenia, Italy, March 28–30, 1988, 158, Litografia Tacchi, Pisa, Italy.Google Scholar). Rationally designed oligopeptides containing an inhibitor of this enzyme showed promising chemotherapeutic effect in the murine model of disseminated candidiasis (14Milewski S. Chmara H. Andruszkiewicz R. Mignini F. Borowski E. Muzzarelli R.A.A. Chitin Enzymology. Alda Tecnografica, Grottamare, Italy1993: 167-176Google Scholar). Moreover, the fungal enzyme is a probable point of regulation of chitin biosynthesis. In the opportunistically pathogenic fungusCandida albicans, activity of this enzyme increases 4–5-fold during yeast-to-mycelia (Y → M) morphological transformation (15Chiew Y.Y. Shepherd M.G. Sullivan P.A. Arch. Microbiol. 1980; 125: 97-104Crossref PubMed Scopus (52) Google Scholar), correlating with a similar change in a chitin content in the cell wall (16Chattaway F.W. Holmes M.R. Barlow A.J.E. J. Gen. Microbiol. 1968; 51: 367-376Crossref PubMed Scopus (157) Google Scholar). This transformation is considered to be a virulence factor during pathogenesis of human tissues (17Lo H.-J. Kohler J.R. DiDomenico B. Loebenger D. Cacciapuoti A. Fink G.R. Cell. 1997; 90: 939-949Abstract Full Text Full Text PDF PubMed Scopus (1521) Google Scholar). The GFA1 gene encoding C. albicans GlcN-6-P synthase has been recently cloned and sequenced (9Smith R.J. Milewski S. Brown A.J.P. Gooday G.W. J. Bacteriol. 1996; 178: 2320-2327Crossref PubMed Google Scholar). In the present communication we describe the results of our further studies concerning overexpression of the GFA1 gene in yeast, purification and characterization of properties of the gene product, aimed especially at regulation of its activity during morphological transformation ofC. albicans cells. Saccharomyces cerevisiae BJ1991 (MATα pep4-3 prb1 ura3 leu2 trp1) was provided by I. Purvis (Glaxo Group Research, Greenwood, UK). C. albicans ATCC 10261 was a gift from M. Payton (GIMB, Geneva, Switzerland). C. albicans and yeast cells were grown in YPD medium (2% glucose, 2% bacterial peptone, 1% yeast extract) at 28 °C with shaking at 200 rpm. C. albicans cells grown overnight in YPD were harvested, washed with saline, and starved overnight in saline at 4 °C. Starved cells were used to inoculate either the YCB/BSA medium containing 1.17% yeast carbon base, 1% glucose, and 0.2% bovine serum albumin or the Lee's medium (18Lee K.L. Buckley H.R. Campbell C.C. Sabouraudia. 1975; 13: 148-153Crossref PubMed Scopus (572) Google Scholar). Yeast form cells grew efficiently in both media at pH 4.5, 28 °C, and Y → M transformation was performed at pH 6.5, 37 °C. Efficiency of the morphological transformation was assessed by cell counting in a Burker chamber. E. coli DH5αF′ (Life Technologies, Inc.) was used for plasmid selection and amplification. Plasmid YEpGW42 (8.7 kb) carrying the S. cerevisiae GFA1 gene on a 3.5-kbEcoRI fragment inserted into YEp352 (19Watzele G. Tanner W. J. Biol. Chem. 1989; 264: 8753-8758Abstract Full Text PDF PubMed Google Scholar) was a gift from W. Tanner (Regensburg, Germany). YEpMA91 was a yeast shuttle vector carrying the LEU2 marker and the promoter and terminator fromPKG1 separated by a BglII site (20Kingsman S.M. Cousens D. Stanway C.A. Chambers A. Wilson M. Kingsman A.J. Methods Enzymol. 1990; 185: 329-341Crossref PubMed Scopus (60) Google Scholar). Standard procedures were used for the isolation and subcloning of plasmid DNA fragments (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Methods for Southern and Northern analyses were the same as cited previously (9Smith R.J. Milewski S. Brown A.J.P. Gooday G.W. J. Bacteriol. 1996; 178: 2320-2327Crossref PubMed Google Scholar). PCR amplification was for 30 cycles (1 min at 94 °C, 2 min at 50 °C, and 3 min at 72 °C) followed by 8 min at 72 °C then cooling to 40 °C. The reaction mix used standard concentrations recommended by Perkin-Elmer. The primers were designed to incorporate BamHI sites at either end of the structural gene, while maintaining an optimum environment around the start codon (22Cigan A.M. Donahue T.F. Gene (Amst.). 1987; 59: 1-8Crossref PubMed Scopus (257) Google Scholar): 5′-oligo, 5′-GAG AAA AAT ggA Tcc TAT TAA Aaa ATG TGT GG-3′; 3′-oligo, 5′-CAG ACA ggA TcCATT TTC ATT ACT CAA CAG-3′. The start codon and stop anti-codon are underlined. Mismatches are in lowercase. S. cerevisiae deletion strains YRSu3-21 and YRSu3-31 were propagated in YPD containing d-glucosamine, 5 mg ml−1. The cells were transformed by the lithium acetate method (23Lauermann K. Curr. Genet. 1991; 20: 1-3Crossref PubMed Scopus (16) Google Scholar). Selection for transformants was for LEU+ on YNB minimal agar plates. YRS-C65 and YRS-C53 transformants were propagated in defined YNB media containing 1% glucose, 0.65% YNB, and appropriate supplements at 50 μg/ml and then transferred to YPD medium. YRSC-65 cells (10 g wet weight) from the overnight culture on YPD were harvested by centrifugation (5,000 × g, 4 °C, 10 min) and washed with cold buffer A (25 mm potassium phosphate buffer, pH 6.8, 1 mm EDTA). Cell paste was mixed with 20 g of alumina and frozen. The mixture was carefully thawed and cells were disrupted by grinding in a mortar. Buffer B (25 mmpotassium phosphate buffer, pH 6.8, 1 mm EDTA, 1 mm DTT) was added in small portions until the cell paste became sticky, and grinding was continued. Cell debris and alumina were spun down (10,000 × g, 4 °C, 5 min). Supernatant was saved, and the solid residue was extracted again with buffer B, followed by centrifugation. Both supernatants were combined and centrifuged (35,000 × g, 4 °C, 45 min). Precipitate was discarded, and supernatant was saved as a crude extract. Solution containing 1% protamine sulfate in buffer B was added dropwise to the crude extract (1 ml per 70 mg of protein present in the crude extract), stirred moderately at 4 °C. The precipitated solid was harvested. Supernatant was discarded, and precipitate was washed with buffer B and then combined with 6 ml of buffer C (0.1 m pyrophosphate buffer, pH 6.8, 1 mm EDTA, 1 mm DTT, 10 mmFru-6-P). This suspension was stirred for 30 min at 4 °C and centrifuged (10,000 × g, 4 °C, 10 min). Precipitate was discarded, and supernatant was saved as a pyrophosphate extract. Pyrophosphate extract containing GlcN-6-P synthase activity was filtered through the 0.22-μm Millipore membrane filter, diluted 1:2 with buffer D (25 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm DTT), and loaded on Mono Q HR 5/5 FPLC column equilibrated with buffer D. The column was washed with 5 ml of buffer D, and elution was performed with a linear 0–0.5m NaCl gradient in buffer D at 1.0 ml min−1. Active fractions were pooled and concentrated by ultrafiltration with Centricon 10 device. The concentrated active fraction from Mono Q was loaded on Superdex 200 HR 10/30 column equilibrated with buffer B containing 0.15 m NaCl. Proteins were eluted with the same buffer at the flow rate of 0.5 ml min−1. Active fractions were pooled. A standard incubation mixture consisted of 10 mm Fru-6-P, 10 mml-glutamine, 1 mm EDTA, 1 mm DTT, 50 mm potassium phosphate buffer, pH 6.8, and appropriately diluted enzyme preparation and inhibitors when necessary. Final concentration of the pure GlcN-6-P synthase was 0.5–1.0 μg ml−1. The reaction was started by adding the enzyme, incubated at 37 °C for 30 min, and terminated by heating at 100 °C for 1 min. The concentration of GlcN-6-P formed by the enzyme, determined by the modified Elson-Morgan procedure (24Kenig M. Vandamme E. Abraham E.P. J. Gen. Microbiol. 1976; 94: 46-54Crossref PubMed Scopus (99) Google Scholar), increased linearly for at least 60 min. One unit of specific activity was defined as an amount of enzyme that catalyzed the formation of 1 μmol of GlcN-6-P min−1 mg protein−1. l-Glutamate formed by GlcN-6-P synthase was determined by coupling with glutamate dehydrogenase, essentially as described by Badet et al. (2Badet B. Vermoote P. Haumont P.Y. Lederer F. LeGoffic F. Biochemistry. 1987; 86: 1940-1948Crossref Scopus (141) Google Scholar). This method was used to confirm the results obtained from kinetic experiments performed at low concentrations of the substrates. C. albicans cells grown in Lee's medium were harvested and suspended in 8.5-ml portions of 0.1 mimidazole/HCl buffer, pH 7.0, containing 0.2 m KCl and 0.1m MgCl2, at cell density 1.2–1.6 × 109 cells ml−1. Aliquots (1.5 ml) composed of toluene/ethanol/Triton X-100, 5:20:2, were added to the cell suspensions, and the mixtures were vortexed for 5 min at room temperature. The cells were washed three times with 50 mmpotassium phosphate buffer, pH 6.8, containing 1 mm EDTA and 1 mm DDT and suspended in the same buffer at 108 cells ml−1. l-Glutamine, 10 mm, and Fru-6-P, 10 mm, and inhibitors when necessary were added, and the suspensions were incubated for 30 min at 37 °C. Cells were removed by centrifugation, and GlcN-6-P concentration was assayed in the supernatant, as described above. Incubation mixtures containing 5 μg of GlcN-6-P synthase, 50 mm potassium phosphate buffer, pH 6.8, 1 mmEDTA, 15 mm Fru-6-P, and inactivators at various concentrations in a total volume of 1 ml were incubated at 25 °C. To follow an inactivation of the enzyme, 200-μl aliquots were withdrawn from the mixtures, applied at the tops of 1-ml columns packed with Sephadex G-25 (equilibrated previously with 50 mm potassium phosphate buffer, pH 6.8), and centrifuged (500 × g, 1 min, 4 °C). Under these conditions the unbound inhibitor was separated from the enzyme, and protein was recovered in clean test tubes. Appropriate effluent aliquots were used for the determination of the residual enzyme activity. GlcN-6-P synthase, 5 μg, was incubated with group-specific reagents under following conditions: (a) with NTCB, IAA, phenylmethylsulfonyl fluoride, and NAI in 50 mm potassium phosphate buffer, pH 6.8, containing 1 mm EDTA; (b) with DEP in 50 mm potassium phosphate buffer, pH 6.0, containing 1 mm EDTA; (c) with BD in 50 mm bicarbonate buffer, pH 8.5; (d) with CMC in 50 mm MES buffer, pH 6.0; (e) with PLP in 50 mm HEPES buffer, pH 6.5, in the dark, followed by 1-h reduction with 20 mm NaBH3CN. All the incubations were run in a total volume of 1 ml, at 25 °C, except that with DEP was run at 4 °C. Aliquots (200 μl) withdrawn from the incubation mixtures at appropriate time intervals were further processed as described above. Chromatofocusing was performed on a Mono P HR 5/5 column. The purified GlcN-6-P synthase (200 μg) was dissolved in 25 mm Bis-Tris-HCl, pH 6.3, as a starting buffer, and a pH 6 to 4 gradient was generated during the elution with 20 ml of Polybuffer 74 (diluted 1:10 in water) solution, pH 4. Samples (0.5 ml) were collected, and pH values and the GlcN-6-P synthase activity were measured. GlcN-6-P synthase, 100 μg ml−1, was preincubated for 0–120 min at 25 °C with 10 μm cAMP, 1 mm ATP, cAMP-dependent protein kinase from beef heart (30 units ml−1), and/or protein kinase inhibitor from rabbit muscle (10 μg ml−1), as indicated. Each sample contained 10 mm EDTA and 40 mm NaF. Samples were assayed for GlcN-6-P synthase activity and sensitivity to 0.5 mm UDP-GlcNAc. GlcN-6-P synthase (10 μg ml−1, 30 pmol) was phosphorylated by incubating with cAMP-dependent protein kinase (5 μg ml−1, 62 pmol), [γ-32P]ATP (0.5 mm, 4 μCi nmol−1), 10 μmcAMP, 10 mm EDTA in a total volume of 800 μl of 50 mm Tris-HCl, pH 7.4, at 25 °C for various times. At time intervals, 2× 50 μl samples of the incubation mixture were collected. One of them was treated with protein kinase inhibitor, 1 μg ml−1, and after appropriate dilution assayed for GlcN-6-P synthase activity. In the second sample, the phosphorylation was stopped by adding Laemmli sample buffer, and the mixture was boiled for 3 min and then subjected to SDS-PAGE. Gels were stained with Coomassie Brilliant Blue, and bands corresponding to GlcN-6-P synthase were cut from the gel and counted for radioactivity in Beckman LS 3801 scintillation counter. Blanks obtained from control mixtures containing all components except protein kinase, processed as described above, were subtracted from the particular values. Gel filtration was performed on Superdex 200 HR 10/30, eluted at 0.5 ml min−1 with 25 mm potassium phosphate buffer, pH 6.8, containing 0.15 m NaCl, 1 mmDTT, and 1 mm EDTA. Protein elution was followed at 280 nm, and GlcN-6-P synthase activity was measured colorimetrically in 0.5-ml samples. Native PAGE was run at 4–9% acrylamide concentration, and the data were treated as described (25Hedrick J.L. Smith A.J. Arch. Biochem. Biophys. 1968; 126: 155-164Crossref PubMed Scopus (1488) Google Scholar). Native GlcN-6-P synthase, 0.5 mg ml−1 in 50 mm triethanolamine buffer, pH 8.0, containing 0.15m KCl, was treated at 25 °C with 0.01% glutaraldehyde for 8 h. SDS was added to 0.6%, the samples were incubated at 37 °C for 1 h and then submitted to SDS-PAGE analysis run in 3% gel with cross-linked rabbit phosphorylase b oligomers as molecular mass markers. Small quantities of yeast or C. albicans cells were broken by the small scale glass beads procedure (26Schatz G. Methods Enzymol. 1976; 56: 40-50Crossref Scopus (6) Google Scholar). Phosphoglucose isomerase activity was assayed according to the procedure of Stein (27Stein M.W. Methods Enzymol. 1968; 1: 299-310Google Scholar) and glutaminase activity by the method of Holcenberg (28Holcenberg J.S. Methods Enzymol. 1985; 113: 257-263Crossref PubMed Scopus (10) Google Scholar). Glucose 6-phosphate concentration was assayed as described by Lowry and Passoneau (29Lowry O.H. Passoneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, NY1972: 72-73Google Scholar). Discontinuous SDS-PAGE was performed by the method of Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) with 5% stacking gel and 7.5% separating gel. Native PAGE was performed as described previously (31Bollag D.M. Edelstein S.J. Protein Methods. Wiley-Liss, NY1992: 143-160Google Scholar). Protein was assayed by the Bradford procedure (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin as a standard. Glutamine analogs were synthesized by Dr. R. Andruszkiewicz, Technical University of Gdańsk. [γ-32P]ATP (4 μCi nmol−1) was from Amersham, UK. Other reagents were from Sigma. EcoRI fragment of the YEpGW42 plasmid containing the S. cerevisiae GFA1 gene was ligated into theEcoRI site of vector pBR325 (6 kb, Life Technologies, Inc.). The backbone of the deletion casette was obtained by band purification of an 8.5-kb fragment after digestion with XhoI andBglII. A 1.1-kb HindIII fragment encoding a functional URA3 gene was obtained by digestion of pSPUR1, band-purified, and ligated into the polylinker of pGEM-7Zf(+) (3,000 base pairs, Promega). Transformants that gave white colonies on isopropyl β-d-thiogalactoside, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside plates were isolated. The 1.1-kb fragment was then excised by digestion with XhoI andBamHI, thus generating URA3 fragment containing ends compatible with the deletion cassette backbone. Ligation yielded plasmid pRS where residues −31 to 971 of GFA1 had been replaced with the URA3 gene. Yeast strain BJ1991 was transformed with the mixture of the restriction fragments excised withSspI, and URA3 colonies were selected. Four were picked and shown to require glucosamine for growth on YPD plates. YRSu3-21 and YRSu-31 were isogenic deletion strains except for the orientation of insertion of the URA3 gene. Northern analysis confirmed that these strains did not produce any mRNA hybridizing to theGFA1 gene (Fig. 1), and no GlcN-6-P synthase activity could be detected. The C. albicans GFA1 gene (9Smith R.J. Milewski S. Brown A.J.P. Gooday G.W. J. Bacteriol. 1996; 178: 2320-2327Crossref PubMed Google Scholar) was amplified by PCR using primers that incorporated BamHI sites 5′ and 3′ to the coding sequence. The PCR 2.1-kb product was digested withBamHI, band-purified, and ligated into the BglII site of pMA91. Restriction mapping was used to determine that obtained plasmids YEpRSC-65 and YEpRSC-53 contained the GFA1 gene in the correct orientation relative to the PGK1 promoter. These plasmids were transformed into strains YRSu3-21 and YRSu3-31. They complemented the glucosamine auxotrophy, restored GlcN-6-P synthase activity, and a GFA1 transcript of the correct size was detected by Northern analysis (Fig. 1). Several isolated YRSC-65 and YRSC-53 transformants when grown in YPD medium reproducibly produced C. albicans GlcN-6-P synthase constituting 5–7% of total cytoplasmic proteins, as revealed by densitometric SDS-PAGE analysis. The C. albicans GlcN-6-P synthase, overproduced by YRSC-65 cells, was purified to at least 97% homogeneity with 52% yield using a four-step procedure involving protamine sulfate precipitation, ion-exchange chromatography, and gel filtration, as summarized in TableI. Two enzymes that could affect the kinetic measurements, namely phosphoglucose isomerase and glutaminase, were not precipitated with protamine sulfate and therefore absent from the pyrophosphate extract and more purified preparations. Such a purification could be completed in 2 days. Isolation from 10 g of wet weight cells afforded 4.4 mg of the electrophoretically homogenous protein. The enzyme was relatively stable in crude extract, when stored at −20 °C; repeated thawing and freezing did not affect its activity for at least 1 month. More purified preparations were stable at −20 °C for weeks when stored in 50% glycerol. The presence of 1 mm DTT and 10 mm Fru-6-P was essential for the enzyme stability. The pure enzyme exhibited pH optimum 6.8 ± 0.05 in Tris, Bis-Tris, HEPES, Mops, and phosphate buffers.K m for l-Gln was 1.56 mm,K m for Fru-6-P was 1.41 mm, andk cat was 1150 min−1. TheK m values were also determined for the enzyme present in crude extracts prepared from C. albicans ATCC 10261 Y and M cells. The former exhibited K m forl-Gln was 1.52 mm and K m for Fru-6-P was 1.45 mm, whereas the latter had the sameK m for Fru-6-P, but the K m forl-Gln was 0.82 mm, and an inhibition of the enzyme activity by an excess of this substrate was observed (details not shown). Substrate inhibition was not detected for the pure enzyme and for the enzyme present in the crude extract from C. albicans Y cells.Table IPurification of C. albicans GlcN-6-P synthase from YRSC-65 cellsVolumeTotal proteinTotal activitySpecific activityRecoveryPurication factormlmgunitsunits mg−1%−foldCrude extract1017540.80.3271001Pyrophosphate extract82934.01.18833.6Mono Q47.421.72.93538.95Superdex 20044.421.24.835214.8 Open table in a new tab Pure GlcN-6-P synthase was chromatofocused using a 6 to 4 pH gradient on MonoP HR 5/5 FPLC column. The enzyme was partially denatured during column development, but an activity profile (not shown) enabled an estimation of an isoelectric point of 4.6 ± 0.05. Molecular weight of theC. albicans GlcN-6-P synthase subunit was determined by SDS-PAGE. From the plot of lg(M r)versus migration distance, M r = 79,500 was obtained, which is in a good agreement with the value 79,482, deduced from the gene sequence. The molecular weight of the native protein was determined by gel filtration and native PAGE run under variety of acrylamide concentrations. The first method gave reproducibly M r = 340,000 and the latterM r = 330,000 (Fig.2). In both methods only single bands were detected. The native enzyme was treated with glutaraldehyde for 8 h. SDS treatment of such a preparation and subsequent separation of its components by SDS-PAGE led to the appearance of bands corresponding toM r = 80,000, 178,000, and 325,000 (not shown). It may be assumed that the bands corresponded to the monomeric, dimeric, and tetrameric forms of the enzyme, respectively. No band corresponding to the possible trimeric form was found. Pure native C. albicans GlcN-6-P synthase was incubated with several chemical reagents under conditions ensuring selective modification of particular amino acid residues. Samples drawn from the reaction mixtures at time intervals were subjected to enforced gel filtration, and activity of GlcN-6-P synthase was determined in effluent aliquots. Incubation with Cys-directed NTCB and IAA, Arg-directed BD, His-directed DEP, Lys-directed PLP, Asp/Glu-directed CMC, and Tyr-directed NAI led to a time- and concentration-dependent irreversible modification of the enzyme, whereas Ser-directed phenylmethylsulfonyl fluoride had no effect. In each case the inactivation was complete at the appropriate reagent concentration. The pattern of the plots of apparent inactivation velocity constants (k app)versus inactivator concentration derived from these experiments were the straight lines originating at zero point (not shown), thus indicating single-step reactions. Kinetic analysis of the results afforded the apparent second-order rate constantsk 1, summarized in TableII. These values were rather low, except that found for DEP. Reaction orders determined from the plots of lg(k app) = n lg([I]) were found to be in the 0.87–1.18 range. The residues modified by the group-specific reagents were not unequivocally identified, but the protective effect of enzyme s