Hydrolase and glycosynthase activity of endo-1 , 3-- glucanase from the thermophile Pyrococcus furiosus

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage.


Introduction
For the biocatalytic production of oligosaccharides, glycoside hydrolases are of interest because of their generally simple heterologous production and the availability of low-cost substrates.Synthesis of oligosaccharides is achieved by thermodynamically controlled reverse hydrolysis and kinetically controlled transglycosylation, which both require high substrate concentrations and low water activity, often achieved by elevated temperatures and the addition of organic solvents.Final yields, however, do not exceed 50% because the product is a substrate of the enzyme's hydrolase activity.Increased yields have been obtained at higher temperatures with thermophilic enzymes (Boon et al. 1998, Kaper et al. 2001).Currently, a maximal yield can be accomplished only by using glycosynthases, glycoside hydrolases in which the catalytic nucleophile has been replaced by a small non-catalytic amino acid residue (Mackenzie et al. 1998).Glycosynthases couple activated glycosyl donors, which have the opposite anomeric configuration to that of the normal substrate, to a range of acceptors.Because these enzymes cannot hydrolyze the product, they carry out transglycosylation efficiently (Figure 1).Typical product yields are 65-80%, but some studies have reported yields of up to 100% (for reviews, see Moracci et al. 2001, Planas and Faijes 2002, Williams and Withers 2002).The glycosynthase strategy has now been applied to five exo-βglycosidases (Mackenzie et al. 1998, Trincone et al. 2000, Nashiru et al. 2001, Jakeman and Withers 2002, Perugino et al. 2003), four endo-β-glucanases (Malet and Planas 1998, Fort et al. 2000, Hrmova et al. 2002, Jahn et al. 2003) and an exo-αglucosidase (Okuyama et al. 2002).The combination of two endo-glycosynthases resulted in the efficient synthesis of hexasaccharides through the sequential transglycosylation of different disaccharides (Faijes et al. 2001).

General
The NMR spectra were recorded on a Varian Gemini-300 spectrometer (Varian, Palo Alto, CA) in deuterium oxide (D 2 O).Chemical shifts (δ in ppm) were referenced to residual DMSO (δ H 2.49) or d 6 DMSO (δ C 39.7) as internal standards for 1 H or 13 C NMR, respectively.Mass spectra were recorded on a VG Autospec spectrometer (Waters-Micromass Technologies, Milford, MA) by the fast atomic bombardment (FAB) technique with an NaCl matrix.

Hydrolytic activity of wild-type and E170A enzymes
Kinetics with laminarin as substrate Reactions were performed with 0.5% laminarin in 100 mM sodium phosphate buffer, pH 6.5, and 0.02 µM wild-type enzyme or 1 µM E170A mutant, at 80 °C.Aliquots of the reaction were withdrawn at 10-min intervals, and the increase in reducing power was deter- mined by the dinitrosalicylic acid (DNS) method (Miller 1959), with glucose as the standard.
Kinetics with 4-methylumbelliferyl glycoside substrates Kinetics with Substrates 1 and 2 were performed by measuring changes in UV absorbance at 365 nm due to the release of 4-methylumbelliferone with a Varian Cary 4 spectrophotometer with a Peltier temperature control system.Rates of enzyme-catalyzed hydrolysis were determined by incubating the substrate (0-8 mM) in phosphate buffer (100 mM, pH 6.5) for 5 min in a thermostated cell holder at 80 °C, followed by addition of the corresponding enzyme (0.02 µM for wild type or 1 µM for E170A) and monitoring the absorbance change at λ = 365 nm (molar extinction coefficient, Δε = 4452 M -1 cm -1 ).

High performance liquid chromatography (HPLC)
Reaction mixtures containing the substrate (4 mM) and wild-type LamA (20-200 nM) in phosphate buffer (100 mM), pH 6.5, were incubated at 80 °C.Aliquots were taken at different time intervals and analyzed by HPLC in a Nova-Pak C18 column (4 µm, 3.9 × 150 mm) (Waters, Milford, MA).The flow rate was 1 ml min -1 , with 16% MeOH in 50 mM phosphate buffer (pH 6.5) as the mobile phase, and detection at 316 nm.Only products containing the 4-methylumbelliferyl chromophore were detected at this wavelength.Yields were calculated from the peak areas based on the corresponding response factor determined for free 4-methylumbelliferone (8770 area mM -1 ) and for 4-methylumbelliferyl glycosides (7603 area mM -1 ).Chromatographic peaks were identified by co-injection with independent standards.

Glycosynthase activity of E170A LamA
α-Laminaribiosyl fluoride (6) (1 mM) and different glycosyl acceptors (5 mM) were dissolved in phosphate buffer (100 mM, pH 6.5) and pre-incubated for 5 min.The E170A mutant (2-7.5 µM) was added and the reactions were incubated at 35, 50 or 76 °C.Aliquots were taken at different time intervals, diluted with H 2 O and analyzed by HPLC as described previously (mobile phase 16-18% MeOH).New chromatographic peaks were first tentatively identified by co-injection with independent standards.Control reactions without enzyme were performed to assess the absence of any uncatalyzed condensation.The thermal stability of α-lamina-ribiosyl fluoride was analyzed in phosphate buffer (100 mM, pH 6.5) by monitoring fluoride anion release with a fluoride selective electrode (Sentek, Essex, U.K.), interfaced with a CiberScan Bench pH/ion meter (Eutech Instruments, Nijkerk, The Netherlands) as previously reported (Faijes et al. 2003).
A second condensation product corresponding to the tetrasaccharide Glcβ3Glcβ4Glcβ3Glcβ-MU was isolated: m/z: calculated for C 34 H 48 O 23 Na: 847.250; found: 847.248. 1 H and 13 C NMR are identical to those reported in Faijes et al. (2003).

Hydrolase activity and specificity of wild-type LamA
The glucoside (5) and the cellobioside (4) were not hydrolyzed by wild-type LamA, whereas the 4-methylumbelliferyl laminaribioside (1) and the mixed-linked oligosaccharides Glcβ4Glcβ3Glcβ-MU (2) and Glcβ4Glcβ4Glcβ3Glcβ-MU (3) were substrates.Cleavage specificity was determined by HPLC (Figures 3A-C).Only chromophoric products (free MU and MU-glycosides) were analyzed (UV detection at 316 nm) and identified by co-injection with independent standards.Reactions were performed at low enzyme concentrations to extend the time course and allow the detection of transient intermediates.
Hydrolysis of 4-methylumbelliferyl laminaribioside (Glcβ3Glcβ-MU) (1) by LamA produced an MU concentration of up to 70% of the initial substrate concentration, and 15% Glcβ-MU (5) was formed after 400 min (10% Glcβ3Glcβ-MU substrate was not hydrolyzed).Therefore, we concluded that the laminaribioside (1) was hydrolyzed at the glycosidic bond with the aglycon and at the β-1,3 bond with a cleavage ratio of 4.5:1.The disaccharide isomer, Glcβ4Glcβ-MU (4), was not hydrolyzed by LamA, indicating that the enzyme does not accept β-1,4 linkages between the glucosyl units in subsites -1 and -2, but accepts β-1,3 linkages in this position.(Enzyme subsites are defined as the set of amino acid residues in the binding site (cleft) that interact with a monosaccharyl unit of the oligomeric substrate, and are numbered as -1, -2, ..., -n, for subsites on the non-reducing end, and +1, +2, ..., +n on the reducing end of the saccharide ligand from the site of cleavage (scissile glycosidic bond).) With the Glcβ4Glcβ3Glcβ-MU (2) substrate (Figure 3B), Glcβ3Glcβ-MU (1) was initially formed (up to 3%), but was then hydrolyzed, and Glc-MU (1%) and MU (85%) were the final hydrolysis products after 400 min of reaction (10% of the substrate was not hydrolyzed).Because Glcβ4Glcβ-MU (4) was not hydrolyzed, the Glcβ-MU formed as the final product was unlikely to have been formed by cleavage of the β-1,3 bond of the trisaccharide, but was likely formed by hydrolysis of the initial disaccharide product 1.Formation of this transient disaccharide indicates that the enzyme can also hydrolyze the β-1,4 glycosidic bond of the β1,4-β-1,3-trisaccharide substrate.Finally, the tetrasaccharide Glcβ4-Glcβ4Glcβ3 -Glcβ-MU (3) was hydrolyzed with the release of MU (Figure 3C), and no other shorter chromophoric oligosaccharides were detected.Even though LamA is an endo-glycosidase, as previously established for the enzyme-catalyzed hydrolysis of β-1,3glucans (Gueguen et al. 1997), the cleavage pattern for Glcβ3Glcβ-MU (1) and Glcβ4Glcβ3Glcβ-MU (2) shows that it has some detectable exo-activity on simple oligosaccharides, with both β-1,4 and β-1,3 specificity.Comparison with other laminarinases indicates that this behavior is unique.However, it should be noted that studies on substrate specificity of laminarinases are not as extensive as those on lichenases from Family 16.LamR from Rhodothermus marinus has endo-activity on laminarin, but shows some exo-activity on oligosaccharides (Petersen et al. 2000, Borriss et al. 2003).Additionally, both β-1,3 and β-1,4 specificities have been reported for LamR; the enzyme hydrolyzes mixed-linked β-1,3-1,4 glucans with β-1,4 glycosidic bonds with higher specific activity than β-1,3 glucans.Moreover, activity on cello-oligosaccharides, but not on polysaccharides, has also been reported (Petersen et al. 2000).Hydrolysis of β-1,4 bonds of carboxymethyl cellulose has been observed with the putative laminarinases GluA and GluC from Lysobacter enzymogenes (Palumbo et al. 2003).However, the lichenase Lic16A from Clostridium thermocellum hydrolyzes lichenan and barley β-glucan, but with β-1,3 cleavage specificity (Fuchs et al. 2003).These examples illustrate that the specificities of laminarinases and lichenases in Family 16 of glycoside hydrolases cover a broad spectrum and require reclassification based on sequence similarity and substrate specificity criteria.
The substrate Glcβ3Glcβ-MU (1) was selected as the reference substrate for wild-type LamA glycosidase activity because it is preferentially hydrolyzed with release of the chromophoric aglycon, MU.The reaction follows Michaelis-Menten kinetics (Figure 4) with k cat = 4.22 ± 1.69 s -1 and K M = 0.93 ± 0.79 mM.

Hydrolase and glycosynthase activity of E170A
Residue Glu170 of Pyrococcus furiosus laminarinase is proposed to act as the catalytic nucleophile (Gueguen et al. 1997), based on sequence similarity with other Family 16 glycosyl hydrolases for which the catalytic residues have been experimentally identified (Juncosa et al. 1994, Viladot et al. 1998, Planas 2000).The Glu170 residue was replaced by alanine to produce the mutant LamA, E170A, to confirm the role of Glu170 as an essential catalytic residue in the hydrolytic mechanism, and to evaluate its potential as a glycosynthase.
The hydrolase activity of the E170A mutant was measured with laminarin and several 4-methylumbelliferyl β-glycosides as substrates, and compared with the hydrolase activity of the wild-type enzyme (Table 1).With laminarin, the E170A mutant retained only 0.05% activity relative to the wild-type enzyme, whereas a 10 4 -fold reduction was obtained with the laminaribioside substrate (1).This effect is consistent with Glu170 being the catalytic nucleophile.
Because a laminaribiosyl unit binds productively in subsite -2/-1 (as deduced from hydrolysis of Glcβ3Glcβ-MU (1) by wild-type LamA), α-laminaribiosyl fluoride (6) was used as the glycosyl donor to determine whether the E170A mutant has glycosynthase activity with different acceptors.Reactions were monitored by HPLC to detect MU-glycoside products.With Glcβ-MU (5) as the acceptor and a reaction temperature of 35 °C, a trisaccharide product (yield ≈ 2% after 3 h reaction) was obtained.When the reaction was performed at 50 °C, the same trisaccharide was produced with a maximum yield of 20% at 24 h (4.5% after 3 h reaction).The yield did not increase with longer incubation or with an increase in temperature to 76 °C, at which the enzyme is probably more active because of its thermophilic nature.The initial rate of condensation was higher at 76 °C (17.7 × 10 -4 s -1 ) than at 50 °C (5.7 × 10 -4 s -1 ), but the yield of condensation product did not exceed 10% at the higher temperature, most likely because of spontaneous hydrolysis of the α-glycosyl fluoride donor (Faijes et al. 2003).The reaction at 50 °C was performed on a semi-preparative scale (10 mg of donor, 0.6 mg enzyme, two equivalents of acceptor) (Table 2).Two main products were isolated after chromatographic separation of the reaction mixture.The first (20% yield, as determined by HPLC) was identified as Glcβ3Glcβ4Glcβ-MU ( 7 Table 1.Hydrolase activity of wild-type and E170A LamA on laminarin and 4-methylumbelliferyl β-glycosides.Reactions took place at 80 °C in 100 mM phosphate buffer, pH 6.5, containing 0.5% laminarin or 4 mM MU-glycoside.Activity was determined as release of the chromophoric aglycon (MU = 4-methylumbelliferone).
In conclusion, LamA hydrolyzed mixed-linked oligosaccharides, confirming that the laminaribiose moiety is the preferred substrate unit; however, the enzyme also showed exo-activity, hydrolyzing both β-1,3 and β-1,4 linkages.The E170A LamA mutant possessed glycosynthase activity, catalyzing the condensation of α-laminaribiosyl fluoride (6) donor with Glcβ-MU (5) and Glcβ3Glcβ-MU (1) as acceptors.The reaction was not regiospecific because both β-1,3 and β-1,4 glycosidic bonds were formed; with the monosaccharide ac-LIESHOUT, FAIJES, NIETO, VAN  ceptor 5, a 7:1 ratio of β-1,4 to β-1,3 was obtained, whereas a 1:17 ratio was observed with the disaccharide acceptor 1.These results contrast those reported for a Family 17 1,3-βglucanase from barley (Hrmova et al. 2002), in which the corresponding glycosynthase mutant (E231A) is regiospecific for β-1,3 glycosidic linkages.Family 16 laminarinases seem to include a wider range of specificities.Transglycosylation catalyzed by wild-type laminarinases from Oerskovia sp. and Spisula sachalinensis exclusively produced β-1,3 linkages (Borriss et al. 2003), whereas LamR from R. marinus catalyzed the synthesis of both β-1,3 and β-1,4 bonds (Krah et al. 1998).The efficiency of E170A LamA as a glycosynthase is rather poor, at least with α-glycosyl fluoride as the activated donor, with preparative yields not exceeding 30% in condensation products (yields not optimized).This is probably a consequence of the hyperthermophilic nature of LamA from Pyrococcus furiosus.The maximum temperature for hydrolase activity of wild-type LamA is 100-105 °C, and activity drops to 10% at 50 °C (Gueguen et al. 1997).The α-glycosyl fluoride donor is labile at high temperatures and is readily hydrolyzed above 50 °C.Therefore, the temperature used in this study is a compromise between donor stability and enzymatic activity that renders conditions far from optimal.