Glycobiology Advance Access originally published online on April 3, 2007
Glycobiology 2007 17(7):744-753; doi:10.1093/glycob/cwm039
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Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 ß-glucosidase glycosynthase mutants
2 Center for Protein Structure and Function, Mahidol University, Bangkok 10400, Thailand
3 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
4 Protein Engineering Network of Centres of Excellence, Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
5 Institute of Science, School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
1 To whom correspondence should be addressed; Tel: +66 02 2015845; Fax: +66 02 2015843; e-mail: scjsv{at}mahidol.ac.th
Received on November 29, 2006; revised on March 20, 2007; accepted on March 25, 2007
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Abstract |
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Rice BGlu1 ß-glucosidase is a glycosyl hydrolase family 1 enzyme that acts as an exoglucanase on ß-(1,4)- and short ß-(1,3)-linked gluco-oligosaccharides. Mutations of BGlu1 ß-glucosidase at glutamate residue 414 of its natural precursor destroyed the enzyme's catalytic activity, but the enzyme could be rescued in the presence of the anionic nucleophiles such as formate and azide, which verifies that this residue is the catalytic nucleophile. The catalytic activities of three candidate mutants, E414G, E414S, and E414A, in the presence of the nucleophiles were compared. The E414G mutant had approximately 25- and 1400-fold higher catalytic efficiency than E414A and E414S, respectively. All three mutants could catalyze the synthesis of mixed length oligosaccharides by transglucosylation, when
-glucosyl fluoride was used as donor and pNP-cellobioside as acceptor. The E414G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than that of E414S and E414A, respectively, and gave yields of up to 70–80% insoluble products with a donor–acceptor ratio of 5:1. 13C-NMR, methylation analysis, and electrospray ionization–mass spectrometry showed that the insoluble products were ß-(1,4)-linked oligomers with a degree of polymerization of 5 to at least 11. The BGlu1 E414G glycosynthase was found to prefer longer chain length oligosaccharides that occupy at least three sugar residue-binding subsites as acceptors for productive transglucosylation. This is the first report of a ß-glucansynthase derived from an exoglycosidase that can produce long-chain cello-oligosaccharides, which likely reflects the extended oligosaccharide-binding site of rice BGlu1 ß-glucosidase. Key words: ß-glucosidase / glycosynthase / oligosaccharide synthesis / rice / transglucosylation
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Introduction |
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TopAbstractIntroductionResultsMaterials and methodsConflict of interest statementReferences |
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The use of glycosynthases, catalytic nucleophile mutants of retaining glycosidases, is a highly efficient approach for the enzymatic synthesis of various glycosides and oligosaccharides (Moracci et al. 2001
; Williams and Withers 2002; Perugino et al. 2005). In this technology, the glycoside hydrolase in which the carboxylate nucleophile has been replaced by a nonnucleophilic amino acid cannot form the glycosyl enzyme intermediate, resulting in a hydrolytically inactive enzyme. However, by the use of glycosyl fluorides of inverted configuration relative to the natural substrate as substrates (Williams and Withers 2000), transglucosylation can be achieved in the presence of suitable glycosyl acceptors, giving reaction products that are not hydrolyzed and accumulate in very high yield.
To date, several glycosynthases have been developed from different families of glycosidases, including both exoglycosidases and endoglycosidases. The exoglycosynthases derived from "exoglycosidases" (Mackenzie et al. 1998; Trincone et al. 2000; Nashiru et al. 2001; Jakeman and Withers 2002; Drone et al. 2005; Faijes et al. 2006; Müllegger et al. 2006) have moderate substrate specificity and regioselectivity, based on that of the original wild-type enzymes, and can catalyze the formation of various glycosidic linkages of short-chain oligosaccharides (di-, tri-, and tetrasaccharides) as their major products. The endoglycosynthases derived from "endoglycosidases" (Malet and Planas 1998; Fort et al. 2000; Hrmova et al. 2002; Jahn et al. 2003; van Lieshout et al. 2004; Kim et al. 2006; Sugimura et al. 2006) have high regioselectivity and catalyze the synthesis of one specific glycosidic linkage. Unlike exoglycosynthases, they use their long glycone-binding site to accommodate longer glycosyl donor substrates, such as -cellobiosyl fluoride or -cellotriosyl fluoride, which can form polymeric or even crystalline polysaccharide products by self-condensation or transfer to suitable acceptors. Glycosynthases were originally derived from retaining ß-glycosidases, but their source was recently expanded to include a retaining -glycosidase. Okuyama et al. (2002) reported an -glycosynthase from Schizosaccharomyces pombe that catalyzed the formation of -glycosidic linkages using ß-glucosyl fluoride as donor and pNP--glucoside as acceptor. Moreover, Honda and Kitaoka (2006) reported a glycosynthase derived from an inverting glycosidase, which hydrolyzes the glycosidic bond via a single displacement mechanism with anomeric inversion. Glycosynthase technology is a versatile tool for synthesizing novel glycosides and oligosaccharides, since the directed evolution of glycosynthases, in which other point mutations within the catalytic site were created, was found to improve catalytic activity and substrate specificity (Kim et al. 2004, 2005; Lin et al. 2004). Moreover, the regioselectivity of glycosidic linkages can be altered by changing the stereochemistry of the acceptor substrate (Stick et al. 2004; Faijes et al. 2006).
Rice (Oryza sativa) BGlu1 ß-glucosidase (E.C. 3.2.1.21
[EC]
) is a glycosyl hydrolase family 1 enzyme which is active on pNP-ß-glycoside substrates, primarily pNP-ß-D-fucoside and pNP-ß-D-glucoside (Opassiri et al. 2003). The BGlu1 enzyme also acts as an exo-ß-glucanase that can hydrolyze gluco-oligosaccharides, with preference for short (1,3)-linked (degree of polymerization, DP, 2–3) and long (1,4)-linked oligosaccharides (DP 3–6) (Opassiri et al. 2004). The native BGlu1 enzyme also showed transglucosylation activity toward those oligosaccharide substrates; however, the newly formed products were obtained in lower yields since they were subsequently hydrolyzed over the course of time (Opassiri et al. 2003).
Because rice ß-glucosidase has a long aglycone-binding site, we were interested to study the exoglucansynthase properties of mutated rice BGlu1 and explore its ability to synthesize long oligomeric saccharides, since many exoglycosynthases had been shown to synthesize only short oligomers of di-, tri-, and tetrasaccharides. In this paper, we confirm the catalytic nucleophile of rice BGlu1 by mutagenesis and rescue, and compare the abilities of mutant rice BGlu1 ß-glucosidases to synthesize cello-oligosaccharides of varying length (DP 3–11). These oligosaccharides were longer than products from other glycosynthases derived from exoglycosidases, perhaps reflecting the long aglycone-binding pocket of this enzyme.
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Results |
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TopAbstractIntroductionResultsMaterials and methodsConflict of interest statementReferences |
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Construction and purification of mutant BGlu1
Based on the alignment of the consensus sequence (ITENG) with other family 1 glycosyl hydrolases for which the catalytic nucleophile was conserved (Opassiri et al. 2003
), E414 of rice BGlu1 ß-glucosidase precursor (E386 of the predicted mature protein) was selected for site-directed mutagenesis. All the mutant BGlu1 genes were subcloned into the pET32a(+) vector and overexpressed in Origami (DE3) Escherichia coli. Four-liter cultures of E. coli were harvested for one-batch purification to yield about 10–20 mg of pure mutant enzyme. However, proteolytic cleavage of the His6-tag from the recombinant BGlu1 was observed during cell lysis, which lowered the purification yield of the enzyme. To improve the yield, both phenylmethylsulfonylfluoride (PMSF) and trypsin inhibitor were included in the extraction buffer. The hydrolytic activities toward the 2,4-dinitrophenyl-ß-D-glucoside (2,4-DNPG) substrate of three mutant enzymes were at least 3000–40 000-fold lower than the wild type (Table I), indicating that E414 is indeed important for catalysis.
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Rescue kinetics
Incubation of the three BGlu1 mutants, E414G, E414S, and E414A, with 2,4-DNPG, with sodium formate or sodium azide in 50 mM sodium acetate buffer (pH 5.0), resulted in increased activity as a function of the concentration of the nucleophile. No rescue activity was observed for any of the three mutant enzymes when incubated with the substrate in acetate buffer alone (data not shown). Saturating kinetic behavior was observed for the plot of initial rate as a function of anionic nucleophile concentration. However, the rescue activity tended to decrease at high concentrations of formate (>4 M) and azide (>1 M). The activity of wild-type enzyme also dramatically decreases in the presence of azide at concentrations >0.25 M (data not shown). The apparent kcat and Km values for each mutant enzyme were obtained by incubating with fixed concentrations of 0.5 M azide and 2 M formate (saturating concentrations) and varying the concentration of 2,4-DNPG. The kinetic parameters for cleavage of 2,4-DNPG are summarized in Table I.
The activity of the E414G mutant can be rescued very well in the presence of the anionic nucleophiles azide and formate: the catalytic efficiencies (kcat/Km) with this mutant were approximately 25- to 1400-fold higher than with the E414A and E414S mutants, as shown in Table I.
When the E414G mutant enzyme was incubated with 2,4-DNPG in the presence of sodium azide, a newly formed product was found by thin-layer chromatography (TLC) (data not shown), which was expected to be -D-glucopyranosyl azide based on the inverting mechanism of a ß-glucosidase enzyme when the nucleophilic catalytic residue has been mutated (Wang et al. 1994). Indeed, when the reaction mixtures were analyzed by 1Proton NMR, 1H-NMR, the -anomeric configuration of the glucosyl azide product (J(H, N3) = 4.4 Hz, 
= 5.4 ppm) was confirmed.
Transglucosylation kinetics
To explore the glycosynthase activities of all mutants, they were incubated with an equimolar ratio of -glucosyl fluoride (-GlcF and pNP-cellobioside (pNPC2) acceptor in 150 mM ammonium bicarbonate buffer (pH 7.0), at 30 °C. The newly formed pNP-oligosaccharide products were observed by TLC for all mutants (Figure 1). The kinetic behavior of this glycosynthase reaction was then studied by measurement of the rate of fluoride ion release, as shown in Figure 2.
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We examined the effects of pH on the glycosynthase reaction by measuring transglucosylation rates at different pH values. The optimum pH for all three mutant enzymes for transglucosylation activity was pH 6 (Figure 3), and all three mutants retained 50% catalytic activity at pH 8–9. Study of the transglucosylation activity of the E414G mutant using buffers with overlapping pH ranges showed that enzyme activity was not affected by the buffering species and was at pH 6.0–6.5 in all buffers (data not shown). Hydrolysis of
-GlcF when incubated with enzyme alone was also detected (dotted line). Sodium phosphate buffer at pH 6.0 was chosen for further study of tranglucosylation kinetics, since the hydrolysis reaction was slow enough at that pH to be reliably subtracted.
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), serine mutant (
). The dotted line (
) shows the hydrolysis rate of the E414G mutant control reaction.The transglucosylation activities of the three mutated enzymes at various concentrations of
-GlcF donor and with a fixed concentration of 20 mM pNPC2 acceptor are compared in Figure 4A and the kinetic parameters are summarized in Table II. The E414G mutant shows the highest transglucosylation efficiency (kcat/Km), which is 2.5-fold higher than that of E414S and 14.5-fold higher than that of E414A.
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), and alanine (
) mutants at different 
), pNPC2 (
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The transglucosylation rates of the E414G mutant with different lengths of pNP-cello-oligosaccharides as acceptor and
-GlcF as donor were determined, as shown in Figure 4B. The catalytic efficiency (kcat/Km) with pNP-cellotrioside (pNPC3) is about 6-fold higher than that with pNPC2. No transglucosylation activity toward pNP-glucoside acceptor (pNPG) was observed.
Oligosaccharide synthesis
Because the E414G mutant had the highest transglucosylation activity, as shown in Table II, this mutant was chosen for oligosaccharide synthesis. The products of equimolar reactions between 10 mM -GlcF donor and 10 mM oligosaccharide acceptors in 150 mM ammonium bicarbonate buffer (pH 7.0), at 30 °C were checked by TLC (Figure 5). When incubated with mutant enzyme, the -GlcF was slowly hydrolyzed to free glucose at a rate somewhat greater than that of spontaneous hydrolysis (Figure 5A, lanes 1 and 2). No transglucosylation products were observed when pNPG was used as an acceptor, as expected (Figure 5A, lanes 3 and 4). Interestingly, the mutant enzyme efficiently catalyzed the synthesis of transglucosylation products (P1–P3) when using pNPC2 acceptor (Figure 5A, lanes 5 and 6). The P1–P3 products were expected to be pNP-tri-, -tetra-, and -pentasaccharide, respectively, and longer pNP-oligosaccharides were seen as a dark spot at the origin. For oligosaccharide acceptors (without a pNP group), no transglucosylation products were detected with cellobiose as the acceptor (Figure 5B, lanes 1 and 2). However, longer oligosaccharide products were observed when cellotriose and cellotetraose were used as acceptors (Figure 5B, lanes 3–6). No products were observed when pNP-maltoside or pNP-N,N'-diacetyl-chitobioside were used as acceptors, consistent with a strong preference for ß-linked gluco-oligosaccharide acceptors.
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Reactions between
-GlcF donor and pNPC2 acceptor with varying molar ratios of donor–acceptor (D:A ratio) were performed. When the -GlcF was completely utilized, the soluble products were characterized by high-performance liquid chromatography (HPLC) and the molecular mass [M + Na]+of each eluted product was confirmed by mass spectrometry. The first HPLC peak eluted was the remaining pNPC2 acceptor (486 m/z) and the following products were pNP-glucotrioside (648 m/z), pNP-glucotetraoside (810 m/z), pNP-glucopentaoside (972 m/z), pNP-glucohexaoside (1134 m/z), and pNP-glucoheptaoside (1296 m/z), respectively (Figure 6). The result shows better accumulation of longer pNP-oligosaccharides with a higher D:A ratio (Figure 6C), while a higher amount of acceptor than donor limited the elongation of products (Figure 6A). Therefore, the DPs of oligosaccharide products can be controlled by the ratio of donor–acceptor. With a 1:1 D:A ratio (Figure 6B), the yields of soluble pNP-oligosaccharides ranging from DP 3 to 7, based on the conversion of pNPC2 acceptor, were 25, 14, 8, 2.5, and 0.2%, respectively. The mutated enzyme was able to catalyze the synthesis of water-insoluble polymeric products with higher donor to acceptor ratios, with a small amount of white flocculent precipitate appearing at a 3:1 D:A ratio. At a 5:1 D:A ratio, the E414G mutant rapidly catalyzed the synthesis of precipitated products, giving 70–80% yields within one night.
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Characterization of glycosidic linkage of oligosaccharide products
The precipitated products from the reactions between
-GlcF and pNPC2 catalyzed by the E414G mutant were first analyzed by digesting with ß-(1,4)-endoglucanase A (Cen A) from Cellulomonas fimi, which specifically hydrolyzes ß-(1,4)-glycosidic bonds (Wong et al. 1986). The reaction products formed over time were analyzed by TLC. The endoglucanase activity of the enzyme releases various aryl-saccharides and short-chain oligosaccharides. The hydrolysis products appeared to be pNPG, pNPC2, pNPC3, cellobioside, and cellotrioside. The results here imply that the synthesis products are cello-oligosaccharides because they can be hydrolyzed by ß-(1,4)-endoglucanase (data not shown).
The 13C-NMR spectrum of the precipitated products of the reaction using -GlcF donor and pNPC2 acceptor catalyzed by the E414G mutant was similar to that of the pNPC3 standard (Figure 7). This indicated that the glycosidic bonds have a ß-(1,4)-linkage, in which the signal of a C4-O-ß-linked glucose appears at around 79–80 ppm (Bock et al. 1984). No peaks were observed at a chemical shift of about 84–86 ppm, where a C3-O-ß-linked glucose would resonate (Bock et al. 1991). Peaks at this chemical shift, characteristic of ß-(1,3)-linked oligosaccharides, had been previously reported for the precipitated products formed by glycosynthase mutants of barley (1,3)-ß-D-glucan endohydrolase (Hrmova et al. 2002) and Bacillus licheniformis endo-(1,3;1,4)-ß-D-glucanase (Faijes et al. 2004).
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The exclusive formation of ß-(1,4)-linkages was confirmed by methylation analysis. Two permethylated alditol acetates (PMAAs) prepared from precipitated products of the reaction between 50 mM
-GlcF and 10 mM pNPC2 catalyzed by the E414G mutant were detected by gas liquid chromatography (GLC) with the same retention times as the PMAAs prepared from cellotetraose. These derivatives were identified by electron impact mass spectroscopy as 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol, derived from (1,4)-D-glucosyl residues, as a major derivative and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol, derived from terminal nonreducing end glucosyl residues, as a minor derivative. This confirmed that the reaction product was exclusively a linear (1,4)-ß-linked oligosaccharide. The average DP of the insoluble products was estimated from the ratio of peak areas between (1,4)-D-glucosyl residues and the terminal nonreducing end residue to be approximately 9 (the ratio of cellotetraose standard was approximately 4).
Analysis of DP length of insoluble products by electron spray ionization–mass spectrometry
The mass spectrum of the precipitated products from the reaction between -GlcF donor and pNPC2 acceptor with a 5:1 D:A ratio shows a distribution of pNP-cello-oligosaccharide products ranging from DP 5 to 11 (Figure 8). The difference between each peak is approximately 162 m/z, which is equivalent to the mass of a glucosyl residue.
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Discussion
The rice BGlu1 ß-glucosidases mutated at E414 had very low hydrolytic activity with 2,4-DNPG substrate, since the carboxylate group of the catalytic nucleophile was not present. To confirm the identity of E414 as the catalytic nucleophile and ensure that the mutant enzymes were correctly folded, the rescue of the glycosidic bond-cleaving activity of each mutant by the anionic nucleophiles azide and formate was demonstrated. These external nucleophiles fit into the space created by removal of the carboxylate of the mutant enzyme and function as exogenous nucleophiles, as shown in Figure 9. The expected result is that the activity of an enzyme mutated at the catalytic nucleophile can be increased many fold in the presence of anionic nucleophiles (Moracci et al. 2001
). The catalytic efficiencies of the three mutants rescued by azide or formate were in the order E414G > E414A > E414S (Table I). Similarly, lower activity of the serine mutant compared with the alanine mutant was also observed in glycosynthases derived from Agrobacterium sp. ß-glucosidase (Mayer et al. 2000) and C. fimi Man2a (Zechel et al. 2003). The bulkier hydroxymethyl group of serine and the methyl group of alanine presumably hinder the attack of the anionic nucleophile on the substrate. The higher rescue activity of the glycine mutant compared with the other mutants was also reported in barley (1,3)-ß-D-glucan endohydrolases glycosynthases (Hrmova et al. 2002).
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The transglucosylation activities of all the mutants were first explored by incubating them with an equimolar ratio of
-GlcF donor and pNPC2 acceptors, and the newly formed pNP-oligosaccharide products were observed by TLC (Figure 1). As can be seen, the glycine mutant has the best transglucosylation activity and completely converted the -GlcF donor to transglycosylation products (P1–P4). The optimum pH for all three mutant enzymes for transglucosylation activity was pH 6.0–6.5, which is about 1 unit higher than the pH optimum (pH 5.0) of the native enzyme, possibly reflecting a lower dependence on acid catalysis for the glycosynthase reaction than for the wild type, as might be expected. Interestingly, the pH-dependence of the hydrolytic reaction tracked that of transglucosylation, suggesting that the hydrolysis observed is indeed (mutant) enzyme-catalyzed, rather than spontaneous. This conclusion is supported by TLC analysis of the reaction products formed over time (data not shown), which revealed faster hydrolysis of the -GlcF donor when incubated with mutant enzymes than in the control (without enzyme). The enzymatic hydrolysis of -GlcF has previously been reported for some glycosynthases (Faijes et al. 2003; van Lieshout et al. 2004; Kim et al. 2006).
The transglucosylation activities of the three mutated rice ß-glucosidases were compared. The Km values for -GlcF of all three mutants were similar, but the kcat values are quite different. The E414G mutant shows the highest transglucosylation rate, with a kcat value 3-fold higher than E414S and 19-fold higher than E414A (Table II). Similar orders of reaction efficiency have been reported for other glycosynthases (Mayer et al. 2001; Hrmova et al. 2002; Jahn et al. 2003). The better transglucosylation activity of the serine mutant compared with the alanine mutant glycosynthase derived from Agrobacterium sp. was explained on the basis of an interaction of the hydroxyl group of serine with the departing fluoride (Mayer et al. 2000). The three dimensional structure of the glycine mutant glycosynthase of ß-mannanase Man26A (Jahn et al. 2003) showed a water molecule in a position similar to that expected for the serine hydroxyl in the serine mutant glycosynthases. It was proposed that the better catalysis by the glycine mutant than the serine mutant might be partially due to the greater freedom of the water molecule to position itself to interact optimally with the departing fluoride than can the more rigid hydroxyl group of the serine side-chain. The interactions in the rice BGlu1 glycosynthases are likely to be similar.
To assess the interaction of the acceptor molecule with the glucose-binding subsites of the substrate-binding cleft, the transglucosylation activities with different acceptors of the E414G mutant were compared (Figure 4B). It appears that the aglycone-binding subsite of the enzyme prefers a longer chain length of pNP-oligosaccharide acceptor, as can be seen by the 4-fold lower Km of pNPC3 compared with the Km of pNPC2. The mutant rice BGlu1 has no transglucosylation activity toward the pNPG acceptor, indicating that the subsite +1 of mutant rice BGlu1 may not productively bind the glucose residue linked to the aromatic ring of the p-nitrophenyl residue, which would need to occupy subsite +2 (see Figure 2). These results correspond to our previous report (Opassiri et al. 2004) that the wild-type enzyme has lower Km for hydrolysis of longer cello-oligosaccharide substrates and that subsite +1 of wild-type BGlu1 showed negative binding affinity toward cello-oligosaccharides (–0.76 ± 0.05 kJ/mol). However, it does not match the transglycosylation activity of the wild-type enzyme, which transglycosylates pNPG to form pNPC2 and pNPC3 products (Opassiri et al. 2004). This suggests that the pNPG acceptor for the glycosynthase reaction competes with -GlcF at the subsite –1, rather than acting as an attacking acceptor to form new product. In contrast, pNPC2 and longer pNP-oligosaccharides act as productive acceptors, most likely due to cooperative binding in subsite +2, which provides the largest contribution of binding affinity (15.7 ± 0.05 kJ/mol), with the other positive binding subsites +3 to +5. Structural studies are currently underway to determine the positions and interactions involved in these subsites (Chuenchor et al. 2006), which should lead to a better understanding of this interaction.
The effectiveness of oligosaccharide synthesis catalyzed by E414G mutant using -GlcF and various oligosaccharide acceptors was studied (Figure 5). With pNPC2 as acceptor, the utilization of -GlcF was complete with less free glucose (G) release by hydrolysis, indicating that the mutant rice ß-glucosidase preferred these pNP-oligosaccharides to water as competitive nucleophilic acceptors. However, the utilization of pNPC2 was incomplete, suggesting that the newly formed products acted as more productive substrates for consecutive transfers. No transglucosylation products were observed when pNPG was used as an acceptor, as expected (see above). Also, pNP-N,N'-diacetyl ß-D-chitobioside and pNP-ß-D-maltoside did not act as acceptors, indicating the importance of the ß-linkage of the sugars and 2-hydroxyl groups on the sugars for productive binding. This is consistent with the acceptor binding to the same glucose subsites demonstrated for hydrolysis of cello-oligosaccharides (Opassiri et al. 2004). Unlike the pNPC2 acceptor, no glycosynthase products were observed when cellobiose was used as acceptor, indicating that the extra binding of the p-nitrophenyl group of pNPC2 at subsite +3 is important for productive binding. The pNP ring has been reported to facilitate the tighter binding of sugar moieties in a cooperative fashion in other glycosynthases (Mackenzie et al. 1998; Faijes et al. 2003; Drone et al. 2005). Furthermore, cellotriose and cellotetraose are better acceptors than cellobiose, as indicated by the longer DPs of oligosaccharide products observed when they were used. This result indicated that the enzyme requires an acceptor that occupies up to subsite +3 for optimal transglucosylation efficiency.
The E414G mutant was capable of synthesizing soluble oligosaccharides of varying length (DP 3–7) with moderate yields, depending on the ratio of D:A (Figure 6). The long-chain oligosaccharide products were suppressed at higher concentrations of acceptor, as would be expected. Our result was different from that with the E358A Abg glycosynthase, which gave only pNP-trisaccharide (79%) and pNP-tetrasaccharide (13%), but no longer oligosaccharides, when an equimolar ratio of the same substrates was used (Mackenzie et al. 1998). These results suggested that the rice mutant enzyme has less preference for pNPC2 than does the Abg mutant, but has a greater preference for longer pNP-oligosaccharide products that become more effective substrates for consecutive transfers. However, when used at a 5:1 D:A ratio the E414G mutant catalyzed the synthesis of precipitated products very quickly, giving 70–80% yields within one night, clearly indicating that the mutated rice BGlu1 prefers to transfer the glucosyl moiety to long oligosaccharide substrates.
Linkage analysis of the precipitated products by digestion with Cen A, 13C-NMR spectroscopy, and methylation confirmed that the mutated rice ß-glucosidase exclusively catalyzed the formation of ß-(1,4) glycosidic linkages. An average DP of 9 was estimated from the methylation analysis of a mixture of pNP-oligosaccharides, consistent with the distribution of DP 5–11 determined by ESI-MS. The cello-oligosaccharide products formed by the rice BGlu1 mutant crystallize when sufficient concentrations of the long-chain pNP-cello-oligosaccharide products of DP >5 are produced. Cellodextrins (cello-oligosaccharides) of DP 
6 have been reported as soluble oligomers in water (Pereira et al. 1988; Zhang and Lynd 2005), while gluco-oligomers having an average DP of about 8 synthesized by an endoglycosynthase (Fort et al. 2000) or by the cellodextrin phosphorylase from Clostridium thermocellum were insoluble (Samain et al. 1995). This is, however, the first report of an exoglycosidase-derived glycosynthase which can catalyze the synthesis of such long-chain oligosaccharides.
In conclusion, we report an "exoglycosynthase" derived from rice BGlu1 ß-glucosidase that exploits its long aglycone-binding subsite to synthesize long-chain oligosaccharides like those previously only reported from "endoglycosynthase"-catalyzed transglycosylations. The synthesized products contained exclusively ß-(1,4) glucosidic linkages and had DPs from 3 to at least 11. The glycine mutant showed the highest transglucosyation activity and catalyzed the rapid synthesis of insoluble polymeric products in up to 80% yields. The transglucosylation kinetics revealed that the rice BGlu1 glycosynthase prefers long-chain oligosaccharide acceptors, which must fill its sugar residue-binding subsites at least up to subsite +3 for reasonable transglucosylation efficiency. These mutated rice ß-glucosidases have potential for large-scale synthesis of cello-oligosaccharides in high yields using a cheap -GlcF donor substrate.
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Materials and methods |
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TopAbstractIntroductionResultsMaterials and methodsConflict of interest statementReferences |
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Cellobiose and p-nitrophenyl ß-glycosides were purchased from Sigma Chemical Co. (St. Louis, MO) Cello-oligosaccharides (DP 3—4) were from Seikagaku Kogyo Co. (Tokyo, Japan).
-GlcF and 2,4-DNPG were synthesized by previously described methods (Sharma et al. 1995; Yokoyama 2000). Cen A from C. fimi was a generous gift from Professor R.A.J. Warren of the University of British Columbia, Vancouver, Canada.
Construction of mutated rice ß-glucosidases E414A, E414S, and E414G
All mutants were constructed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Forward and reverse primers, respectively, were as follows: 5'-G ACA GTC GTC ATA ACT GCA AAA CGG AAT GGA TCA AC-3' and 5'-G TTG ATC CAT TCC GTT TGC AGT TAT GAC GAC TGT C-3' for mutant E414A; 5'-AT CCG ACA GTC GTC ATA ACT AGC AAC GGA ATG GAT CAA CCT GC-3' and 5'-GC AGG TTG ATC CAT TCC GTT GCT AGT TAT GAC GAC TGT CGG AT-3' for mutant E414S; and 5'-G ACA GTC GTC ATA ACT GGG AAC GGA ATG GAT CAA C-3' and 5'-G TTG ATC CAT TCC GTT CCC AGT TAT GAC GAC TGT C-3' for mutant E414G. The template DNA consisted of the wild-type rice ß-glucosidase (BGlu1) cDNA with SnaB I and EcoR I sites introduced at the 5' and 3' ends, respectively, inserted in the EcoR V site of pBlueScript II SK(+) (Opassiri et al. 2003). All mutants were verified by complete sequencing of the insert gene.
Protein expression and purification
All three mutants were subcloned in between the EcoR V and Eco RI site of pET32a(+), transformed into Origami (DE3) E. coli and expressed as previously described (Opassiri et al. 2003). The isopropyl ß-D-1-thiogalactopyranoside induced cultures were scaled up to 4 L to attain high amounts of mutant enzymes. For protein purification, the cell pellets were lysed in extraction buffer, containing 20 mM Tris–HCl (pH 8.0), 1% Triton-X 100, 200 µg/mL lysozyme, 1 mM PMSF, and 0.1 mg/mL trypsin inhibitor from soybean (Sigma Co.) to reduce proteolytic degradation of mutant enzymes. The cell suspension was incubated at room temperature for 30 min. The cell suspensions were sonicated on ice, cell debris was removed by centrifugation (12 000 g, 10 min) and the protein was purified from the soluble cell extract on a Co2 + affinity column (5 mL HiTrap Chelating HP, Amersham Biosciences). The column was equilibrated with 5 column volumes of equilibration buffer (20 mM Tris–HCl (pH 8.0), 150 mM NaCl), then washed with 10 column volumes of wash buffer (20 mM imidazole in equilibration buffer), before elution of bound proteins with 250 mM imidazole in the same buffer. Imidazole was removed by buffer exchange with 50 mM sodium phosphate buffer (pH 6.0), in a 10 kDa centrifugal filter (Amicon, Billerica, MA). The protein was concentrated to a 1 mL final volume, and further purified by cation exchange chromatography on an SP sepharose Fast Flow column (HiTrap SP FF 5 mL, Amersham Biosciences, Piscataway, NJ). The column was equilibrated with 5 column volumes of 50 mM phosphate buffer (pH 6.0). Proteins were eluted with a 0–250 mM NaCl gradient in the same buffer at a flow rate of 1.0 mL/min. Fractions with protein purity higher than 95%, as judged by sodium dodecyl sulfate–polacrylamide gel electrophoresis, were pooled, and the buffer was exchanged with 20 mM phosphate buffer (pH 6.0), and the pool concentrated using a 10 kDa centrifugal filter (Amicon). Protein concentration was determined by measuring absorbance at 280 nm. An 
280 of 113560 M–1 cm–1, derived by the method of Gill and von Hippel (1989), was used to calculate the enzyme concentration of both wild type and mutants of rice BGlu1.
Rescue kinetics
Stock solutions of 5 M sodium azide and sodium formate in 50 mM sodium acetate buffer (pH 5.0), were prepared immediately prior to use. All three mutant enzymes and wild-type BGlu1 were assayed under standard conditions (50 mM sodium acetate buffer (pH 5.0), 30 °C) with the 2,4-DNPG substrate. The release of 2,4-dinitrophenol was measured by absorbance at 400 nm in a Cary 4000 spectrophotometer (Varian Inc., Palo Alto, CA). The extinction coefficient for 2,4-dinitrophenol at 400 nm (
= 9545 M–1 cm–1) was determined by measuring the absorbance of carefully prepared stock solutions in 50 mM sodium acetate buffer (pH 5.0), at 30 °C. Spontaneous hydrolysis of 2,4-DNPG was negligible at all concentrations.
Kinetic analyzes of transglucosylation reactions
Kinetic parameters of mutated enzymes were determined at 30 °C by incubating 20-120 µM enzymes in 150 mM sodium phosphate buffer (pH 6.0), in the presence of -GlcF donor and pNP-cello-oligosaccharide acceptor. Initial rates of fluoride release were determined using an Orion fluoride electrode (model 96-09BN, interfaced with a Fischer Scientific Accument 925 pH/ion meter). Sodium fluoride was used as a standard. Kinetic parameters were determined by nonlinear regression analysis with Graphpad Prism 3.02 software.
The pH optima were determined over the range of 4–9 in 150 mM buffers as follows: sodium acetate (pH 4–5); sodium phosphate (pH 6–7); and ammonium bicarbonate (pH 8–9). For the E414G mutant, the pH optimum was further verified with overlapping buffer ranges, as follows: sodium acetate (pH 4.0–6.5), sodium phosphate, (pH 5.0–8.0), and ammonium bicarbonate (pH 6.5–8.0). The reaction was performed by incubating 20–120 µM purified mutant enzyme with 50 mM -GlcF donor and 10 mM pNPC2 acceptor in different pH buffers. All transglucosylation rates were corrected for the hydrolysis rate of the control reactions without the acceptor substrate.
Oligosaccharide synthesis
The 20–40 µM E414G mutant was incubated with 10–100 mM concentrations of -GlcF and acceptor glycoside (10–30 mM) in 150 mM ammonium bicarbonate buffer (pH 7.0), at 30 °C for 16 h. Reactions were monitored by TLC (silica gel 60 F254, aluminum-backed, Merck, Darmstadt, Germany) using 7:2.5:1 ethyl acetate (EtOAc)–methanol (MeOH)–water as solvent for pNP-oligosaccharides and 2:1:1 EtOAc–acetic acid (HOAc)–water for oligosaccharides. Plates were visualized by exposure to 10% sulfuric acid in ethanol followed by charring. The reaction mixture was centrifuged at 13 000 g for 5 min. The precipitate was collected, thoroughly washed with water, freeze-dried to yield a white powder and kept dry for further characterization. The mutant enzyme in the supernatant was removed by centrifugal filtration (Microcon YM-10). The soluble products were kept at 4 °C.
Characterization of soluble products by HPLC
The soluble products from the reactions between -GlcF donor and pNPC2 acceptor were characterized. After removal of the mutant enzyme by centrifugal filtration, 10 µL of the reaction products were loaded onto a polymer-based reverse phase HPLC column (Prevail carbohydrate ES 5U column, Altech, 150 mm x 4.6 mm) with a Waters 717 HPLC. The column was eluted with a linear gradient from 80:20 to 50:50 acetonitrile/water over 40 min at a flow rate of 0.5 mL/min. The eluted peaks were detected at 300 nm using an ultraviolet (UV)-visible detector. Reaction yields were determined by integration of the product peaks within the HPLC profile. The eluates were collected and the molecular masses of the eluted products were determined by mass spectrometry.
Total carbohydrate assay
Total carbohydrate in the precipitated products was determined by a modification of the phenol–sulfuric acid method (Kacc 1995). The precipitate was suspended in 1 mL water and mixed with 1 mL of 5% phenol reagent (w/v). Five milliliters of concentrated sulfuric acid was added and the samples were cooled at room temperature for 10 min. Total amounts of carbohydrate in the reaction products and starting material substrate (control without enzyme) were determined by comparing the absorbance at 490 nm with a glucose standard curve. Total yield (%) was calculated as the total amount of carbohydrate in precipitated products divided by the total amount in the control reaction multiplied by 100%.
Analysis of DP of insoluble products by ESI–MS
The synthesis reaction was performed in 150 mM ammonium bicarbonate buffer (pH 7.0), containing 50–100 mM -GlcF donor and 10 mM pNPC2 acceptor. The white flocculent precipitate was collected, washed with water, and freeze-dried as described above. A 10–50 mg sample was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.5% trifluoroacetic acid (TFA) in acetonitrile. The final concentration of DMSO was less than 10 mg/L prior to injection to ESI–MS (DMSO/TFA ratio of 1:500 or less). Molecular weights of insoluble products were analyzed by ESI–MS (Perkin Elmer API 300, Sciex, Thornhill, Ontario, Canada). All spectra were obtained in positive-ion, single quadrupole scan mode. The quadrupole mass analyzer was scanned over a range of 500–2500 m/z.
13C-NMR spectroscopy
The white powder of precipitated pNP-oligosaccharides was dissolved in [2H] DMSO at a concentration of 5–10 mg/mL. 13C-NMR spectra were recorded with a Bruker AV 600 spectrometer operated at 298 K and 150.9 MHz. The central peak of the [2H] DMSO multiplet (39.51 ppm) was used as a reference. pNPC3 was used as a standard.
Methylation analysis
PMAAs were prepared from the glycosynthase products (1–2 mg) following the method of Kim et al. (2006). D-Glucitol was used as an internal standard. Briefly, the sample was methylated in the presence of NaOH and CH3I, the glycosidic bonds were hydrolyzed with TFA, and the methylated monosaccharides were reduced with an excess of sodium borohydride and then acetylated with acetic anhydride. The PMAAs were separated and analyzed by GLC–MS (Agilent 6890 N gas chromatograph/5973 N mass selective detector) using an HP-5 capillary column. The derivatives corresponding to the GLC peaks were identified by MS in the electron-impact ionization mode scanning from 50 to 500 m/z. PMAA standards were prepared from cellotetraose and laminaritriose.
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Conflict of interest statement |
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TopAbstractIntroductionResultsMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank the Thailand Research Fund and Mahidol University for financial support. J.S. is a Senior Research Scholar of the Thailand Research Fund (TRF), and J.K.C. is also supported by the TRF. G.H. and W.C. received financial support from the Royal Golden Jubilee PhD Program (Grants No. PHD/0001/2546 and PHD/0113/2546) of the TRF.
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Abbreviations |
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2,4-DNPG, 2,4-dinitrophenyl-ß-D-glucoside; Cen A, ß-(1,4)-endoglucanase A; DMSO, dimethyl sulfoxide; DP, degree of polymerization; ESI–MS, electrospray ionization–mass spectrometry; GLC–MS, gas liquid chromatography–mass spectrometry; HPLC, high- performance liquid chromatography; PMSF, phenylmethylsulfonylfluoride; pNPC2, pNP-ß-D-cellobioside; pNPC3, pNP-ß-D-cellotrioside; pNPC4, pNP-ß-D-cellotetraoside; pNPC5, pNP-ß-D-cellopentaoside; pNPC6, pNP-ß-D-cellohexaoside; pNPC7, pNP-ß-D-celloheptaoside; pNPG, pNP-ß-D-glucoside; TFA, trifluoroacetic acid; TLC, thin-layer chromatography;
-GlcF, -glucosyl fluoride. |
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