Biotechnol Lett DOI 10.1007/s10529-014-1561-y
ORIGINAL RESEARCH PAPER
Characterization and expression of glucosamine-6phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris Sheng Wang • Piwu Li • Jing Su • Xiangkun Wu Rongrong Liang
•
Received: 9 April 2014 / Accepted: 15 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml-1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein -1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The Km and Vmax of the GlcN-6-P synthase towards D-fructose 6-phosphate were 2.8 mM and 6.9 lmol min-1 mg-1, respectively. Keywords Glucosamine-6-phosphate synthase Pichia pastoris Saccharomyces cerevisiae
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1561-y) contains supplementary material, which is available to authorized users. S. Wang P. Li (&) J. Su X. Wu R. Liang Shandong Provincial Key Laboratory of Microbial Engineering, School of Food and Bioengineering, Qilu University of Technology, Jinan 250353, China e-mail: [email protected] S. Wang e-mail: [email protected]
Introduction Glucosamine-6-phosphate (GlcN-6-P) synthase, also known as L-glutamine: D-fructose 6-phosphate amidotransferase (EC 2.6.1.16), is a key enzyme in the pathway of amino sugar biosynthesis. GlcN-6-P synthase in mammals is thought to induce insulin resistance in diabetes (Hebert et al. 1996). Furthermore, fungal GlcN-6-P synthase is considered to be a potential target for antifungal therapy (Borowski 2000). For these reasons, investigation of microbial GlcN-6-P synthase is of some significance. Studies on Escherichia coli and Candida albicans have shown that GlcN-6-P synthase belongs to the L-glutaminedependent aminotransferase (GAT) family, which catalyzes transference of the ammonia moiety of L-glutamine to the corresponding site of D-fructose 6-phosphate. The reaction is as follows: l-glutamine þ d-fructose 6-phosphate ! d-glucosamine 6-phosphate þ l-glutamate This reaction is the rate-limiting step in hexosamine biosynthesis, leading to generation of uridine 50 -diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), which is a precursor of macromolecules, including chitin and mannoprotein in fungi, peptidoglycan and lipopolysaccharide in bacteria, and glycoproteins in mammals (Durand et al. 2008). Prokaryotic GlcN-6-P synthase has been characterized intensively (Teplyakov et al. 2002). E. coliderived GlcN-6-P synthase has been purified and
123
Biotechnol Lett
biochemical and X-ray diffraction analyses have further revealed the biochemical and physical properties of the enzyme (Badet et al. 1987; Teplyakov et al. 2001). However, little is known about eukaryotic GlcN-6-P synthase. Extraction and purification of GlcN-6-P synthase from eukaryotes is a difficult task because of the low levels at which it is typically present. Therefore, it is necessary to overexpress the GlcN-6-P synthase gene in order to accumulate the enzyme. This approach has been used with C. albicans (Sachadyn et al. 2000); however, low yields of the enzyme were obtained. In the present study, we describe the characterization and expression of GlcN-6-P synthase from Saccharomyces cerevisiae in Pichia pastoris for the first time.
Materials and methods Materials Saccharomyces cerevisiae was purchased from the China Center of Industrial Culture Collection (CICC 32919) in Beijing. E. coli DH5a and pEASY-T1 (Transgen Biotech, Beijing, China) were used to clone and sequence the gene encoding GlcN-6-P synthase, GFA1 (GeneBank ID: 853757). P. pastoris host strain SMD1168 and the pPICZaA vector were used to construct an expression system for GlcN-6-P synthase. BMGY and BMMY culture media were prepared according to the Pichia Expression Kit Manual (Invitrogen, Carlsbad, CA, USA). Cloning of the GFA1 Saccharomyces cerevisiae genomic DNA was extracted and purified using the Yeast Genomic DNA Extract Kit (Sangon Biotech, Shanghai, China), which was used as a template for amplifying GFA1, using gene-specific primers. Forward primer, Fp, and reverse primer, Rp, were synthesized by Shanghai Sangon, and EcoRI and SacII restriction sites were engineered into the primer sequences (see Supplementary Table 1). The PCR product was purified and cloned into pEASY-T1 vector and transformed into E. coli DH5a. Positive clones were identified and verified by sequencing pEASY-T1-GFA1 with M13
123
primers (Transgen Biotech, Beijing, China) and comparison with the GFA1 sequence database. Construction of expression vector Verified recombinant pEASY-T1-GFA1 was digested with EcoRI and SacII, and the released fragment was then ligated into EcoRI and SacII sites of the expression vector pPICZaA, generating pPICZaAGFA1. Recombinant pPICZaA-GFA1 was identified with restriction endonuclease cleavage and PCR amplification.
Transformation and expression pPICZaA-GFA1 was linearized with SacI and transformed into P. pastoris host strain SMD1168 by electroporation, using a Gene Pulser apparatus (BioRad, Berkeley, CA, USA). Transformants were inoculated into 25 ml BMGY medium in 250 ml flasks and cultured at 30 °C with shaking at 240 rpm until the OD600 reached 2–5. The cells were harvested by centrifugation at 5,0009g for 5 min and then resuspended into 50 ml BMMY medium in 500 ml flasks. GFA1 expression was induced at 30 °C for 120 h with methanol at 0.5 % (v/v) added at 24 h intervals.
Purification of GlcN-6-P synthase Determination of GlcN-6-P synthase activity A reaction mixture (1 ml) containing 20 mM D-fructose 6-phosphate, 15 mM L-glutamine, 2.5 mM EDTA, and 0.2 ml enzyme was prepared in 25 mM potassium phosphate buffer, pH 7. The mixture was placed at 37 °C for 30 min, and the reaction was terminated by heating to 100 °C for 4 min. GlcN-6-P concentration was determined by the modified colorimetric method of Elson and Morgan (Deng et al. 2005). One unit of activity was defined as an amount of the enzyme that catalyzed formation of 1 lmol GlcN-6-P per min (Badet et al. 1987; Rodriguez-Diaz et al. 2012). Aliquots of the sample solution obtained from crude BMMY medium using different purification procedures were subjected to protein concentration determination using a nanophotometer (Implen, Munich, Germany). SDS-PAGE was performed using a 10 % (v/v)
Biotechnol Lett Table 1 Purification of S. cerevisiae GlcN-6-P synthase from culture medium of P. pastoris SMD1168 (pPICZaA-GFA1) Treatment
Total protein (mg)
Total activity (U)
Specific activity (U mg-1)
Recovery (%)
Purification factor
Crude broth
57.5
75.4
1.31
100
1
Ammonium sulfate
20.3
64.2
3.16
85
2.4
Dialysis Superdex 200
15.6 8.6
60.4 42.1
3.87 4.93
94 70
2.95 3.75
Culture medium was centrifuged at 5,0009g for 5 min; 40 ml was treated with ammonium sulfate at 30–80 % saturation. The precipitate was dissolved in 5 ml 25 mM K2HPO4/KH2PO4 buffer (pH 7) and concentrated to 1 ml by overnight dialysis (MD 44 nm, MW 8000–14000; Solarbio, MA, USA) against PEG 4000. The concentrated solution was applied onto a Superdex 200 column on an AKTA Explorer System followed by elution with 25 mM K2HPO4/KH2PO4 buffer (pH 7) at 0.2 ml min-1. Aliquots of eluant (2 ml) were used for determination of protein concentration and GlcN-6-P synthase activity
separating gel and 5 % (v/v) stacking gel, according to the standard method. Determination of optimal pH and temperature The optimal pH of purified GlcN-6-P synthase was determined at 37 °C and from pH 2 to 8 with 25 mM potassium phosphate buffer. Optimal temperature was assayed at the optimal pH from 25 to 65 °C. Determination of kinetic parameters The reaction rate (lmol min-1 mg-1) of the GlcN-6-P synthase was assayed under the determined optimal conditions (pH 5.5, 55 °C for 50 min), and with a -1 D-fructose-6-phosphate from 1 to 10 mg ml . The reaction rate versus substrate concentration was plotted to verify the reaction mode of the GlcN-6-P synthase according to the Michaelis–Menten equation. Kinetic parameters (Km, Vmax) were graphically determined by Lineweaver–Burk plotting.
Results and discussion Transformation and expression The recombinant pPICZaA-GFA1 vector was constructed by ligation of the pPICZaA vector, containing the a-factor signal sequence, to the gene of interest, which had been linearized with SacI, and transformed into P. pastoris host strain SMD1168 by electroporation. P. pastoris transformants were inoculated onto YPD plates containing zeocin (100, 200, 300, and 400 lg ml-1) to screen for transformants carrying multiple copies. Ten multiple-copy transformants
Fig. 1 GlcN-6-P synthase activity and cell growth of recombinant P. pastoris SMD1168 (pPICZaA-GFA1) in shaking flask culture. Cells and supernatants were harvested every 24 h after induction with 0.5 % (v/v) methanol, and checked for dry cell weight and GlcN-6-P synthase activity. Filled square GlcN-6-P synthase activity. Open square Dry cell weight. The values (mean ± standard deviation) represent three independent experiments
were selected on YPD plates containing 400 lg zeocin ml-1. Genomic DNA of the transformants was extracted, and integration of GFA1 into the P. pastoris genome was confirmed by PCR analysis, using 50 a-factor-specific and 30 AOX1-specific primers. The genomic DNA of P. pastoris SMD1168 with pPICZaA (no insert) was used as control. Agarose gel electrophoresis showed that GFA1 had been integrated into the P. pastoris genome. After being induced with methanol, GlcN-6-P synthase was expressed and secreted into the culture medium. The dry cell weight of the selected transformant reached 26 mg ml-1 and GlcN-6-P synthase activity reached 96 U ml-1 after induction for 120 h (Fig. 1).
123
Biotechnol Lett
Fig. 2 A molecular model of S. cerevisiae GlcN-6-P synthase was predicted using I-TASSER, based on the crystal structure of E. coli GlcN-6-P synthase (PDB: 4AMV) and C. albicans GlcN6-P synthase (PDB: 2POC). The 3-D structure consists of 2 domains, including 16 a-helices and 17 b-sheets. The figure was generated using Pymol software
Analysis of primary structure and 3-D structure The expressed GlcN-6-P synthase was comprised of 717 amino acids, with a molecular weight of 80,047 Da, and a theoretical pI of 6. The amino acid sequence also showed a high degree of homology (83 %) to the corresponding sequence of Zygosaccharomyces bailii ISA1307. The total number of negatively charged residues (Asp ? Glu) and positively charged residues (Arg ? Lys) were 93 and 81, respectively. The instability index (II) was 38.54, which showed that the protein is stable. A molecular model of the 3-D structure of GlcN-6P synthase from S. cerevisiae was constructed on the basis of the crystal structures of E. coli GlcN-6-P synthase (PDB: 4AMV) and C. albicans GlcN-6-P synthase (PDB: 2POC; Fig. 2). The protein is composed of two synthase domains. Like other N-terminal nucleophile hydrolases, it has a terminal glutamine amidotransferase type 2 domain (residues 2–318). The SIS domain (residues 390–529; 562–707), a phosphosugar-binding domain, plays a key role in catalysis and communication of fructose 6-phosphate and GlcN-6-P between the two active sites. Purification of GlcN-6-P synthase The purity of GlcN-6-P synthase of S. cerevisiae in the cultured supernatant of recombinant P. pastoris
123
Fig. 3 SDS-PAGE analysis of S. cerevisiae GlcN-6-P synthase. The gel was stained with Coomassie blue. Lanes 1 and 2 Eluates obtained from a Superdex 200 column on an AKTA Explorer System with 25 mM K2HPO4–KH2PO4 buffer (pH 7) at a flow rate of 0.2 ml min-1. Lanes 3–6 aliquots obtained by dialysis against PEG 4000. Lane 7 culture medium from P. pastoris SMD1168 that had been transformed with pPICZaAGFA1. Lane 8 culture medium from P. pastoris SMD1168 that had been transformed with pPICZaA-no insert. Lane 9 molecular weight marker. The bands corresponding to S. cerevisiae GlcN-6-P synthase is indicated by the arrow
SMD1168 exceeded 90 % (Fig. 3, lane 7); thus, our approach will greatly facilitate the purification of S. cerevisiae GlcN-6-P synthase and decrease the industrial production and application costs. The GlcN-6-P synthase was purified to homogeneity by ammonium sulfate precipitation, dialysis, and Superdex 200 gel filtration. The maximum specific activity of the purified GlcN6-P synthase toward GlcN was 4.93 U mg-1 (Table 1), which was higher than that of the GlcN-6-P synthase purified from Sporothix schenckii (0.78 U mg-1) and from C. albicans (0.21 U mg-1) (Gonzalez-Ibarra et al. 2010; Olchowy et al. 2006), but was lower than that of GlcN-6-P synthase muteins containing internal hexaHis fragments (6.8 U mg-1) obtained from C. albicans (Czarnecka et al. 2012). SDS-PAGE analysis showed one single protein band with an apparent molecular mass of about 90 kDa (Fig. 3), which is larger than the theoretical mass (80,047 Da). This could be because P. pastoris enables some post-translation modifications, including assembly of disulfide bonds, exclusion of signal peptides, and glycosylation. (Chen et al. 2010). Prediction of N-glycosylation and O-glycosylation indicated that GlcN-6-P synthase has no N-linked glycosylation, but has ten potential O-glycosylation sites (Ser and Thr residues) in the C-terminus. Thus, the increased molecular weight of the GlcN-6-P synthase may be attributed to O-linked glycosylation, which did not reduce the activity of GlcN-6-P synthase.
Biotechnol Lett
Fig. 4 Effects of pH and temperature on the GlcN-6-P synthase activity. a pH optimum and stability. The optimal pH of the GlcN6-P synthase was measured at 37 °C and at a pH range of 2–8 with 25 mM potassium phosphate buffer. To confirm stability at various pH values, purified GlcN-6-P synthase was incubated at 37 °C for 1 h at various pH values (potassium phosphate buffer pH: 2–8), and then used to determine the enzyme activity. pH stability was defined as the pH range over which the enzyme activity retained more than 80 % of the original activity. The original activity at the optimum pH of 5.5 was taken as 100 %. Filled square GlcN-6-P synthase relative activity. Open square GlcN-6-P synthase activity. b Temperature optimum and
stability. The optimal temperature of the GlcN-6-P synthase was determined at pH 5.5 and from 25 to 65 °C. The thermostability of GlcN-6-P synthase was determined by incubating it at various temperatures (25–65 °C) for 1 h, and then the enzyme activity was measured in a 25 mM potassium phosphate buffer at pH 5.5. Thermostability was from 25 to 65 °C, over which the activity of the enzyme retained over 80 % of the original activity. The original activity at the optimum temperature of 55 °C was taken as 100 %. Filled circle GlcN-6-P synthase relative activity. Open circle GlcN-6-P synthase activity. The values (mean ± standard deviation) represent three independent experiments
Enzymatic properties of GlcN-6-P synthase
mechanisms of the enzyme, and structural analysis and molecular modification of this protein.
The purified GlcN-6-P synthase was incubated at 37 °C for 1 h at various pH values; it was stable from pH 4.5 to 5.5, retaining more than 80 % of the original activity (Fig. 4a). The optimal temperature for GlcN6-P synthase activity at pH 5.5 was 55 °C at which the highest specific activity was 6.82 U mg protein-1. When preincubated at pH 5.5 for 1 h at various temperatures (25–65 °C), the enzyme retained more than 80 % of its activity at 45–60 °C, but declined above 60 °C. It retained 96–70 % of its original activity at 55 °C (Fig. 4b). Thus, GlcN-6-P synthase demonstrated moderate thermostability and pH stability. The Km and Vmax of the purified GlcN-6-P synthase for D-fructose 6-phosphate were determined from Lineweaver–Burk plots to be 2.8 mM and 6.9 lmol min-1 mg-1, respectively. In conclusion, we cloned the sequence of GFA1 from S. cerevisiae, GlcN-6-P synthase gene, performed its expression in P. pastoris, purified the GlcN6-P synthase to electrophoretical homogeneity, and characterized its enzymatic properties. Our study has significance for further research on the metabolic
Acknowledgments This work was financially supported by the National High-tech R&D Program of China (863 Program, No. 2012AA021504) and the Taishan Scholar Program of Shandong.
References Badet B, Vermoote P, Haumont PY et al (1987) Glucosamine synthease from Escherichia coli: purification, properties and glutamine-utilizing site location. Biochemistry 26:1940–1948 Borowski E (2000) Novel approaches in the rational design of antifungal agents of low toxicity. Farmaco 55:206–208 Chen XZ, Xu SQ, Zhu MS et al (2010) Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris. Process Biochem 45:75–80 Czarnecka J, Kwiatkowska K, Gabriel I et al (2012) Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification. J Mol Recognit 25:564–570 Deng MD, Severson DK, Grund AD et al (2005) Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metab Eng 7: 201–214
123
Biotechnol Lett Durand P, Golinelli-Pimpaneau B, Mouilleron S et al (2008) Highlights of glucosamine-6 P synthase catalysis. Arch Biochem Biophys 474:302–317 Gonzalez-Ibarra J, Milewski S, Villago´mez-Castro JC et al (2010) Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target. Med Mycol 48:110–121 Hebert LF, Daniels MC, Zhou J et al (1996) Overexpression of glutamine: fructose 6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest 98:930–936 Olchowy J, Kur K, Sachadyn P et al (2006) Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli. Protein Exp Purif 46:309–315
123
Rodriguez-Diaz J, Rubio-Del-Campo A, Yebra MJ (2012) Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine. Biotechnol Bioeng 109:1704–1712 Sachadyn P, Jedrzejczak R, Milewski S et al (2000) Purification to homogeneity of Candida albicans glucosamine-6phosphate synthase overexpressed in Escherichia coli. Protein Exp Purif 19:343–349 Teplyakov A, Obmolova G, Badet B et al (2001) Channeling of ammonia in glucosamine-6-phosphate synthase. J Mol Biol 313:1093–1102 Teplyakov A, Leriche C, Obmolova G et al (2002) From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase. Nat Prod Rep 19:60–69
ORIGINAL RESEARCH PAPER
Characterization and expression of glucosamine-6phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris Sheng Wang • Piwu Li • Jing Su • Xiangkun Wu Rongrong Liang
•
Received: 9 April 2014 / Accepted: 15 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml-1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein -1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The Km and Vmax of the GlcN-6-P synthase towards D-fructose 6-phosphate were 2.8 mM and 6.9 lmol min-1 mg-1, respectively. Keywords Glucosamine-6-phosphate synthase Pichia pastoris Saccharomyces cerevisiae
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1561-y) contains supplementary material, which is available to authorized users. S. Wang P. Li (&) J. Su X. Wu R. Liang Shandong Provincial Key Laboratory of Microbial Engineering, School of Food and Bioengineering, Qilu University of Technology, Jinan 250353, China e-mail: [email protected] S. Wang e-mail: [email protected]
Introduction Glucosamine-6-phosphate (GlcN-6-P) synthase, also known as L-glutamine: D-fructose 6-phosphate amidotransferase (EC 2.6.1.16), is a key enzyme in the pathway of amino sugar biosynthesis. GlcN-6-P synthase in mammals is thought to induce insulin resistance in diabetes (Hebert et al. 1996). Furthermore, fungal GlcN-6-P synthase is considered to be a potential target for antifungal therapy (Borowski 2000). For these reasons, investigation of microbial GlcN-6-P synthase is of some significance. Studies on Escherichia coli and Candida albicans have shown that GlcN-6-P synthase belongs to the L-glutaminedependent aminotransferase (GAT) family, which catalyzes transference of the ammonia moiety of L-glutamine to the corresponding site of D-fructose 6-phosphate. The reaction is as follows: l-glutamine þ d-fructose 6-phosphate ! d-glucosamine 6-phosphate þ l-glutamate This reaction is the rate-limiting step in hexosamine biosynthesis, leading to generation of uridine 50 -diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), which is a precursor of macromolecules, including chitin and mannoprotein in fungi, peptidoglycan and lipopolysaccharide in bacteria, and glycoproteins in mammals (Durand et al. 2008). Prokaryotic GlcN-6-P synthase has been characterized intensively (Teplyakov et al. 2002). E. coliderived GlcN-6-P synthase has been purified and
123
Biotechnol Lett
biochemical and X-ray diffraction analyses have further revealed the biochemical and physical properties of the enzyme (Badet et al. 1987; Teplyakov et al. 2001). However, little is known about eukaryotic GlcN-6-P synthase. Extraction and purification of GlcN-6-P synthase from eukaryotes is a difficult task because of the low levels at which it is typically present. Therefore, it is necessary to overexpress the GlcN-6-P synthase gene in order to accumulate the enzyme. This approach has been used with C. albicans (Sachadyn et al. 2000); however, low yields of the enzyme were obtained. In the present study, we describe the characterization and expression of GlcN-6-P synthase from Saccharomyces cerevisiae in Pichia pastoris for the first time.
Materials and methods Materials Saccharomyces cerevisiae was purchased from the China Center of Industrial Culture Collection (CICC 32919) in Beijing. E. coli DH5a and pEASY-T1 (Transgen Biotech, Beijing, China) were used to clone and sequence the gene encoding GlcN-6-P synthase, GFA1 (GeneBank ID: 853757). P. pastoris host strain SMD1168 and the pPICZaA vector were used to construct an expression system for GlcN-6-P synthase. BMGY and BMMY culture media were prepared according to the Pichia Expression Kit Manual (Invitrogen, Carlsbad, CA, USA). Cloning of the GFA1 Saccharomyces cerevisiae genomic DNA was extracted and purified using the Yeast Genomic DNA Extract Kit (Sangon Biotech, Shanghai, China), which was used as a template for amplifying GFA1, using gene-specific primers. Forward primer, Fp, and reverse primer, Rp, were synthesized by Shanghai Sangon, and EcoRI and SacII restriction sites were engineered into the primer sequences (see Supplementary Table 1). The PCR product was purified and cloned into pEASY-T1 vector and transformed into E. coli DH5a. Positive clones were identified and verified by sequencing pEASY-T1-GFA1 with M13
123
primers (Transgen Biotech, Beijing, China) and comparison with the GFA1 sequence database. Construction of expression vector Verified recombinant pEASY-T1-GFA1 was digested with EcoRI and SacII, and the released fragment was then ligated into EcoRI and SacII sites of the expression vector pPICZaA, generating pPICZaAGFA1. Recombinant pPICZaA-GFA1 was identified with restriction endonuclease cleavage and PCR amplification.
Transformation and expression pPICZaA-GFA1 was linearized with SacI and transformed into P. pastoris host strain SMD1168 by electroporation, using a Gene Pulser apparatus (BioRad, Berkeley, CA, USA). Transformants were inoculated into 25 ml BMGY medium in 250 ml flasks and cultured at 30 °C with shaking at 240 rpm until the OD600 reached 2–5. The cells were harvested by centrifugation at 5,0009g for 5 min and then resuspended into 50 ml BMMY medium in 500 ml flasks. GFA1 expression was induced at 30 °C for 120 h with methanol at 0.5 % (v/v) added at 24 h intervals.
Purification of GlcN-6-P synthase Determination of GlcN-6-P synthase activity A reaction mixture (1 ml) containing 20 mM D-fructose 6-phosphate, 15 mM L-glutamine, 2.5 mM EDTA, and 0.2 ml enzyme was prepared in 25 mM potassium phosphate buffer, pH 7. The mixture was placed at 37 °C for 30 min, and the reaction was terminated by heating to 100 °C for 4 min. GlcN-6-P concentration was determined by the modified colorimetric method of Elson and Morgan (Deng et al. 2005). One unit of activity was defined as an amount of the enzyme that catalyzed formation of 1 lmol GlcN-6-P per min (Badet et al. 1987; Rodriguez-Diaz et al. 2012). Aliquots of the sample solution obtained from crude BMMY medium using different purification procedures were subjected to protein concentration determination using a nanophotometer (Implen, Munich, Germany). SDS-PAGE was performed using a 10 % (v/v)
Biotechnol Lett Table 1 Purification of S. cerevisiae GlcN-6-P synthase from culture medium of P. pastoris SMD1168 (pPICZaA-GFA1) Treatment
Total protein (mg)
Total activity (U)
Specific activity (U mg-1)
Recovery (%)
Purification factor
Crude broth
57.5
75.4
1.31
100
1
Ammonium sulfate
20.3
64.2
3.16
85
2.4
Dialysis Superdex 200
15.6 8.6
60.4 42.1
3.87 4.93
94 70
2.95 3.75
Culture medium was centrifuged at 5,0009g for 5 min; 40 ml was treated with ammonium sulfate at 30–80 % saturation. The precipitate was dissolved in 5 ml 25 mM K2HPO4/KH2PO4 buffer (pH 7) and concentrated to 1 ml by overnight dialysis (MD 44 nm, MW 8000–14000; Solarbio, MA, USA) against PEG 4000. The concentrated solution was applied onto a Superdex 200 column on an AKTA Explorer System followed by elution with 25 mM K2HPO4/KH2PO4 buffer (pH 7) at 0.2 ml min-1. Aliquots of eluant (2 ml) were used for determination of protein concentration and GlcN-6-P synthase activity
separating gel and 5 % (v/v) stacking gel, according to the standard method. Determination of optimal pH and temperature The optimal pH of purified GlcN-6-P synthase was determined at 37 °C and from pH 2 to 8 with 25 mM potassium phosphate buffer. Optimal temperature was assayed at the optimal pH from 25 to 65 °C. Determination of kinetic parameters The reaction rate (lmol min-1 mg-1) of the GlcN-6-P synthase was assayed under the determined optimal conditions (pH 5.5, 55 °C for 50 min), and with a -1 D-fructose-6-phosphate from 1 to 10 mg ml . The reaction rate versus substrate concentration was plotted to verify the reaction mode of the GlcN-6-P synthase according to the Michaelis–Menten equation. Kinetic parameters (Km, Vmax) were graphically determined by Lineweaver–Burk plotting.
Results and discussion Transformation and expression The recombinant pPICZaA-GFA1 vector was constructed by ligation of the pPICZaA vector, containing the a-factor signal sequence, to the gene of interest, which had been linearized with SacI, and transformed into P. pastoris host strain SMD1168 by electroporation. P. pastoris transformants were inoculated onto YPD plates containing zeocin (100, 200, 300, and 400 lg ml-1) to screen for transformants carrying multiple copies. Ten multiple-copy transformants
Fig. 1 GlcN-6-P synthase activity and cell growth of recombinant P. pastoris SMD1168 (pPICZaA-GFA1) in shaking flask culture. Cells and supernatants were harvested every 24 h after induction with 0.5 % (v/v) methanol, and checked for dry cell weight and GlcN-6-P synthase activity. Filled square GlcN-6-P synthase activity. Open square Dry cell weight. The values (mean ± standard deviation) represent three independent experiments
were selected on YPD plates containing 400 lg zeocin ml-1. Genomic DNA of the transformants was extracted, and integration of GFA1 into the P. pastoris genome was confirmed by PCR analysis, using 50 a-factor-specific and 30 AOX1-specific primers. The genomic DNA of P. pastoris SMD1168 with pPICZaA (no insert) was used as control. Agarose gel electrophoresis showed that GFA1 had been integrated into the P. pastoris genome. After being induced with methanol, GlcN-6-P synthase was expressed and secreted into the culture medium. The dry cell weight of the selected transformant reached 26 mg ml-1 and GlcN-6-P synthase activity reached 96 U ml-1 after induction for 120 h (Fig. 1).
123
Biotechnol Lett
Fig. 2 A molecular model of S. cerevisiae GlcN-6-P synthase was predicted using I-TASSER, based on the crystal structure of E. coli GlcN-6-P synthase (PDB: 4AMV) and C. albicans GlcN6-P synthase (PDB: 2POC). The 3-D structure consists of 2 domains, including 16 a-helices and 17 b-sheets. The figure was generated using Pymol software
Analysis of primary structure and 3-D structure The expressed GlcN-6-P synthase was comprised of 717 amino acids, with a molecular weight of 80,047 Da, and a theoretical pI of 6. The amino acid sequence also showed a high degree of homology (83 %) to the corresponding sequence of Zygosaccharomyces bailii ISA1307. The total number of negatively charged residues (Asp ? Glu) and positively charged residues (Arg ? Lys) were 93 and 81, respectively. The instability index (II) was 38.54, which showed that the protein is stable. A molecular model of the 3-D structure of GlcN-6P synthase from S. cerevisiae was constructed on the basis of the crystal structures of E. coli GlcN-6-P synthase (PDB: 4AMV) and C. albicans GlcN-6-P synthase (PDB: 2POC; Fig. 2). The protein is composed of two synthase domains. Like other N-terminal nucleophile hydrolases, it has a terminal glutamine amidotransferase type 2 domain (residues 2–318). The SIS domain (residues 390–529; 562–707), a phosphosugar-binding domain, plays a key role in catalysis and communication of fructose 6-phosphate and GlcN-6-P between the two active sites. Purification of GlcN-6-P synthase The purity of GlcN-6-P synthase of S. cerevisiae in the cultured supernatant of recombinant P. pastoris
123
Fig. 3 SDS-PAGE analysis of S. cerevisiae GlcN-6-P synthase. The gel was stained with Coomassie blue. Lanes 1 and 2 Eluates obtained from a Superdex 200 column on an AKTA Explorer System with 25 mM K2HPO4–KH2PO4 buffer (pH 7) at a flow rate of 0.2 ml min-1. Lanes 3–6 aliquots obtained by dialysis against PEG 4000. Lane 7 culture medium from P. pastoris SMD1168 that had been transformed with pPICZaAGFA1. Lane 8 culture medium from P. pastoris SMD1168 that had been transformed with pPICZaA-no insert. Lane 9 molecular weight marker. The bands corresponding to S. cerevisiae GlcN-6-P synthase is indicated by the arrow
SMD1168 exceeded 90 % (Fig. 3, lane 7); thus, our approach will greatly facilitate the purification of S. cerevisiae GlcN-6-P synthase and decrease the industrial production and application costs. The GlcN-6-P synthase was purified to homogeneity by ammonium sulfate precipitation, dialysis, and Superdex 200 gel filtration. The maximum specific activity of the purified GlcN6-P synthase toward GlcN was 4.93 U mg-1 (Table 1), which was higher than that of the GlcN-6-P synthase purified from Sporothix schenckii (0.78 U mg-1) and from C. albicans (0.21 U mg-1) (Gonzalez-Ibarra et al. 2010; Olchowy et al. 2006), but was lower than that of GlcN-6-P synthase muteins containing internal hexaHis fragments (6.8 U mg-1) obtained from C. albicans (Czarnecka et al. 2012). SDS-PAGE analysis showed one single protein band with an apparent molecular mass of about 90 kDa (Fig. 3), which is larger than the theoretical mass (80,047 Da). This could be because P. pastoris enables some post-translation modifications, including assembly of disulfide bonds, exclusion of signal peptides, and glycosylation. (Chen et al. 2010). Prediction of N-glycosylation and O-glycosylation indicated that GlcN-6-P synthase has no N-linked glycosylation, but has ten potential O-glycosylation sites (Ser and Thr residues) in the C-terminus. Thus, the increased molecular weight of the GlcN-6-P synthase may be attributed to O-linked glycosylation, which did not reduce the activity of GlcN-6-P synthase.
Biotechnol Lett
Fig. 4 Effects of pH and temperature on the GlcN-6-P synthase activity. a pH optimum and stability. The optimal pH of the GlcN6-P synthase was measured at 37 °C and at a pH range of 2–8 with 25 mM potassium phosphate buffer. To confirm stability at various pH values, purified GlcN-6-P synthase was incubated at 37 °C for 1 h at various pH values (potassium phosphate buffer pH: 2–8), and then used to determine the enzyme activity. pH stability was defined as the pH range over which the enzyme activity retained more than 80 % of the original activity. The original activity at the optimum pH of 5.5 was taken as 100 %. Filled square GlcN-6-P synthase relative activity. Open square GlcN-6-P synthase activity. b Temperature optimum and
stability. The optimal temperature of the GlcN-6-P synthase was determined at pH 5.5 and from 25 to 65 °C. The thermostability of GlcN-6-P synthase was determined by incubating it at various temperatures (25–65 °C) for 1 h, and then the enzyme activity was measured in a 25 mM potassium phosphate buffer at pH 5.5. Thermostability was from 25 to 65 °C, over which the activity of the enzyme retained over 80 % of the original activity. The original activity at the optimum temperature of 55 °C was taken as 100 %. Filled circle GlcN-6-P synthase relative activity. Open circle GlcN-6-P synthase activity. The values (mean ± standard deviation) represent three independent experiments
Enzymatic properties of GlcN-6-P synthase
mechanisms of the enzyme, and structural analysis and molecular modification of this protein.
The purified GlcN-6-P synthase was incubated at 37 °C for 1 h at various pH values; it was stable from pH 4.5 to 5.5, retaining more than 80 % of the original activity (Fig. 4a). The optimal temperature for GlcN6-P synthase activity at pH 5.5 was 55 °C at which the highest specific activity was 6.82 U mg protein-1. When preincubated at pH 5.5 for 1 h at various temperatures (25–65 °C), the enzyme retained more than 80 % of its activity at 45–60 °C, but declined above 60 °C. It retained 96–70 % of its original activity at 55 °C (Fig. 4b). Thus, GlcN-6-P synthase demonstrated moderate thermostability and pH stability. The Km and Vmax of the purified GlcN-6-P synthase for D-fructose 6-phosphate were determined from Lineweaver–Burk plots to be 2.8 mM and 6.9 lmol min-1 mg-1, respectively. In conclusion, we cloned the sequence of GFA1 from S. cerevisiae, GlcN-6-P synthase gene, performed its expression in P. pastoris, purified the GlcN6-P synthase to electrophoretical homogeneity, and characterized its enzymatic properties. Our study has significance for further research on the metabolic
Acknowledgments This work was financially supported by the National High-tech R&D Program of China (863 Program, No. 2012AA021504) and the Taishan Scholar Program of Shandong.
References Badet B, Vermoote P, Haumont PY et al (1987) Glucosamine synthease from Escherichia coli: purification, properties and glutamine-utilizing site location. Biochemistry 26:1940–1948 Borowski E (2000) Novel approaches in the rational design of antifungal agents of low toxicity. Farmaco 55:206–208 Chen XZ, Xu SQ, Zhu MS et al (2010) Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris. Process Biochem 45:75–80 Czarnecka J, Kwiatkowska K, Gabriel I et al (2012) Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification. J Mol Recognit 25:564–570 Deng MD, Severson DK, Grund AD et al (2005) Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metab Eng 7: 201–214
123
Biotechnol Lett Durand P, Golinelli-Pimpaneau B, Mouilleron S et al (2008) Highlights of glucosamine-6 P synthase catalysis. Arch Biochem Biophys 474:302–317 Gonzalez-Ibarra J, Milewski S, Villago´mez-Castro JC et al (2010) Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target. Med Mycol 48:110–121 Hebert LF, Daniels MC, Zhou J et al (1996) Overexpression of glutamine: fructose 6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest 98:930–936 Olchowy J, Kur K, Sachadyn P et al (2006) Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli. Protein Exp Purif 46:309–315
123
Rodriguez-Diaz J, Rubio-Del-Campo A, Yebra MJ (2012) Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine. Biotechnol Bioeng 109:1704–1712 Sachadyn P, Jedrzejczak R, Milewski S et al (2000) Purification to homogeneity of Candida albicans glucosamine-6phosphate synthase overexpressed in Escherichia coli. Protein Exp Purif 19:343–349 Teplyakov A, Obmolova G, Badet B et al (2001) Channeling of ammonia in glucosamine-6-phosphate synthase. J Mol Biol 313:1093–1102 Teplyakov A, Leriche C, Obmolova G et al (2002) From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase. Nat Prod Rep 19:60–69
