Engineering of a thioglycoligase: randomized mutagenesis of the acid–base residue leads to the identification of improved catalysts

Johannes Müllegger1, Michael Jahn1, Hong-Ming Chen1, R.Antony J. Warren2 and Stephen G. Withers1,3

Protein Engineering Network of Centres of Excellence, 1Department of Chemistry and 2Department of Microbiology, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

3 To whom correspondence should be addressed. E-mail: withers{at}chem.ubc.ca


    Abstract
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 Abstract
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 Materials and methods
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 References
 
Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid–base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid–base mutant of Agrobacterium sp. ß-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid–base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.

Keywords: enzymatic oligosaccharide synthesis/ß-glucosidase/thioglycoligase/thioglycoside


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The chemical synthesis of specific oligosaccharides is challenging, largely because very similar reactivity of all the sugar hydroxyls demands laborious, time-consuming protection and deprotection strategies to provide regiochemical control. Furthermore, stereochemical outcome is also a continuous challenge. An alternative to chemical synthesis of carbohydrates is to use enzymes and currently two different approaches are commonly used. One involves the use of glycosyltransferases, whereas the other exploits the transglycosylation ability of glycoside-degrading enzymes (Koeller and Wong, 2000Go). Recently very helpful variations have been added to the latter approach through the use of mutant enzymes, namely glycosynthases (Mackenzie et al., 1998Go), thioglycoligases (Jahn et al., 2003Go) and thioglycosynthases (Jahn et al., 2004Go). These approaches were developed first using ß-glucosidase from Agrobacterium sp. (Abg), an enzyme belonging to the glycosyl hydrolase (GH) family 1 (Coutinho and Henrissat, 1999Go). Abg is a retaining exoglucosidase and hydrolyzes ß-glucosidic bonds via a two-step mechanism involving the formation and subsequent hydrolysis of a glycosyl enzyme intermediate. The detailed reaction mechanism of this enzyme has been uncovered over the past decade and was summarized in a recent review (Zechel and Withers, 2000Go). The first step of the reaction involves glycosylation of the enzyme, whereby the residue functioning as nucleophile attacks the anomeric center of the substrate glycoside, forming a covalent glycosyl enzyme intermediate. Concomitantly the acid–base catalyst donates a proton to the glycosidic oxygen to neutralize the negative charge forming on the leaving group and facilitate its departure. In a second step the same acid–base catalyst then acts as a base, deprotonating and thereby activating an incoming acceptor molecule. This can be water, in which case hydrolysis results, but can also be a sugar moiety, when transglycosylation is the outcome (Figure 1A).



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Fig. 1. Reaction mechanism of Abg. (A) Glycoside hydrolysis or transglycosylation catalyzed by Abg wt. (B) Abg E170Q-catalyzed thioglycoligase reaction.

 
Thioglycoligases are retaining glycosidases with a mutant acid–base catalyst, allowing them to transglycosylate an activated sugar moiety onto a thio sugar acceptor. Use of a donor sugar with a highly activated leaving group that does not need protonation allows the formation of the glycosyl enzyme: the good leaving group chemically complements the missing catalyst. However, the second step, deglycosylation, is very slow in the absence of base catalysis, unless an acceptor sugar with a thiol at the attacking position is employed. Again, the highly nucleophilic thiol(ate) chemically complements the missing base catalyst (Figure 1B).

Glycosynthases are a different group of mutant glycosidases in which the nucleophile has been ablated. The mutants are able to transglycosylate glycosyl fluoride donors of the opposite anomeric configuration to that of the natural substrate, onto an acceptor sugar. Initially the E358A mutant was described (Mackenzie et al., 1998Go), but in subsequent work it was shown that E358S and E358G are even better catalysts (Mayer et al., 2000Go, 2001Go). In order to screen for better glycosynthase variants, an in vivo screen was developed that used an endoglucanase to cleave the fluorogenic reaction product of the synthase but not the unreacted acceptor, thus facilitating identification of active mutants and making it possible to distinguish different levels of activity fairly easy. This screen was recently successfully employed in a directed evolution approach to generate a much more efficient glycosynthase (Kim et al., 2004Go). Similar results have been obtained with retaining glycosidases from other GH families that were converted into glycosynthases and comprehensive reviews thereof are available (Williams and Withers, 2002Go; Jahn and Withers, 2003Go; Perugino et al., 2004Go). Finally, a combination of these two approaches has been developed in which a double mutant of Abg missing both catalytically active residues has been shown to link successfully {alpha}-glucosyl fluorides onto thiol-containing acceptor sugars. The enzyme itself is completely devoid of any hydrolytic activity and serves primarily as a reaction scaffold (Jahn et al., 2004Go).

Thioglycosides have proved useful as competitive inhibitors of glycosidases (Legler, 1990Go), in the formation of stable complexes for X-ray crystallography (Driguez, 2001Go) and as affinity resins for protein purification (Orgeret et al., 1992Go). Furthermore, their use as non-degradable ligands for lectins is under investigation (Witczak, 1999Go; Dey and Witczak, 2003Go). They therefore represent promising candidates for biotechnological and therapeutic applications. Synthesis of this class of molecules up to now has been achieved via a number of chemical routes (Driguez, 1997Go). Recently, however, through use of thioglycoligases (Jahn et al., 2003Go) and glycosyltransferases (Rich et al., 2004Go), additional synthetic routes have been added. Although naturally occurring S-glucosyltransferases from cruciferous plants have now been cloned and characterized, these enzymes specifically catalyze the formation of glucosinolates and do not have more general applications (Marillia et al., 2001Go).

Here we further investigated the potential of Abg thioglycoligase and probed some of the mechanistic details of the reaction. To do so we developed a coupled assay-based screen, produced a mutant enzyme library in which the acid base catalyst E170 has been subjected to saturation mutagenesis and from this isolated several mutants that are able to perform the thioglycoligase reaction. To overcome some limitations in our screen we developed glucosyl azide as a donor and used this in a coupled assay based on that used in the screen developed to carry out a basic kinetic characterization of the isolated mutants. One of these, Abg E170Q, surprisingly has a kcat ~100-fold higher than that of any other mutant. The best mutants were used in enzymatic syntheses using different combinations of donor and acceptor sugars to explore the synthetic potential of the thioglycoligase approach.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Growth media components were purchased from Difco or EM Science/Merck. Unless noted otherwise, reagents were purchased from Sigma or Bio-Rad; DNA modifying enzymes were obtained from Fermentas or New England Biolabs. Oligonucleotide synthesis and DNA sequencing were carried out by the NAPS unit, UBC.

Creation of the AbgE170X library

pETAbgH6wt (Mayer et al., 2000Go), which is pET29b(+) containing Agrobacterium sp. ß-glucosidase fused to a C-terminal His6 tag, was used as a template in a four-primer mutagenic polymerase chain reaction. Primers used were Abg_mut_fw (GTG ATG TCG GCG ATA TAG GCG CC), AbgE170X_rv (GTT GAA GGT CGC AAC CGC ATC) and AbgE170X_fw (GAT GCG GTT GCG ACC TTC AAC NNN CCT TGG TGC) and T7term (GCT AGT TAT TGC TCA GCG G) for the first reactions. Products of these first reactions and primers Abg_mut_fw and T7term were used for the second reaction. The resulting fragment was cut with XbaI/XhoI and cloned into the original plasmid from which the insert had been removed using the same enzymes. The product of the ligation reaction was transformed into DH5{alpha} and plasmid DNA was prepared directly from about 10 000 colonies washed off the selection plates to give the library pETAbgH6E170X.

Expression and purification of endoglucanase1 and Abg mutants

Endoglucanase 1 (EG1) from Fusarium oxysporum sp. lycopersi was cloned into pPIC9 (Invitrogen) containing an N-terminal {alpha}-factor signal sequence. The plasmid was linearized with BstEI and transformed into Pichia pastoris GS115 according to the Invitrogen manual. Clones growing on a His-deficient medium were tested for expression of endoglucanase activity. All transformants showed similar levels of activity and were further selected for the Mut+ or MutS phenotype. One of the Mut+ clones was finally chosen for large-scale expression of EG1. Briefly, a small preculture was grown overnight at 30°C in YPD medium and used to innoculate eight baffled 2 l flasks, each containing 500 ml of YPD medium. Cells were grown for 24 h covered only with cheese cloth, then centrifuged and resuspended in 3 l of BMM medium. Induction was maintained by adding methanol to a concentration of 0.5% every 24 h and vigorous shaking at 30°C for 3 days. Cells were removed by centrifugation and the supernatant was filtered through a Millipore Steritop filter device (0.22 µm). Subsequently, the supernatant was concentrated in a Millipore pressure cell attached to a 3 l kettle over a polyether sulfone membrane (PM10) with a 10 kDa cutoff pore size. The supernatant was further concentrated on Pall spin filters (10 kDa cutoff), adjusted to 1 M ammonium sulfate and loaded on to a phenyl-Sepharose column. EG1 was found mainly in the flowthrough with the rest of the protein recovered from initial fractions obtained upon elution with decreasing ammonium sulfate concentration. The flowthrough and initial elution fractions were concentrated and the buffer exchanged to 50 mM potassium phosphate pH 7.0 on an Amersham PD10 column. His6-tagged mutants of Abg were expressed and purified essentially as described (Mayer et al., 2000Go). Concentrations of Abg mutants were determined spectrophotometrically at 280 nm using an extinction coefficient of 102 490 M–1 cm–1.

Synthesis of donor and acceptor substrates

2,4-Dinitrophenyl ß-D-glucopyranoside (dNP-Glc), ß-D-glucopyranosyl azide (GlcN3) (Ying and Gervay-Hague, 2003Go) and 4-methylumbelliferyl 4-deoxy-4-thio-ß-D-glucopyranoside (4SGlcMU) (Jahn et al., 2004Go) were synthesized according to literature procedures. Other thio sugars used as acceptors were synthesized by different routes and will be described elsewhere.

Screening of the library

The pETAbgH6E170X library was transformed into electro-competent BL21-DE3 cells and spread on kanamycin-containing LB agar plates. Single colonies were picked and grown in 200 µl of LB–kanamycin medium in round-bottomed 96-well plates overnight at 37°C. Cultures were induced by adding isopropyl ß-D-thiogalactoside to a final concentration of 0.1 mM for 4 h at 30°C. Next the microtiter plates were centrifuged, the supernatants removed and cell pellets lysed in 20 µl of BugBuster reagent containing Benzonase (Novagen). Aliquots of 10 µl of the lysates were mixed with 20 µl of 33 mM dNP-Glc and a saturated concentration of 4SGlcMU in 20% DMF, 50 mM potassium phosphate pH 7.0 and reacted overnight at room temperature. To monitor the reaction, products were separated by thin-layer chromatography (TLC) using ethyl acetate–methanol–water (7:2:1) as eluent. Plates were analyzed under UV illumination and by charring with 10% H2SO4 in methanol. For the second, coupled screen, carried out in the fluorescence plate reader, cell lysates were prepared in the same way and then mixed with 20 µl of 5 mM GlcN3 in a saturated solution of 4SGlcMU (~1.4 mM) in 50 mM potassium phosphate pH 7.0; 1 µl of EG1 (1 mg/ml) was added as screening enzyme (Figure 2). Development of fluorescence was observed in black 96-well plates (Corning) in a fluorescence plate reader (Wallac 1420 VICTOR3, Perkin-Elmer) over 4 h at room temperature.



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Fig. 2. Schematic drawing of the coupled assay used for screening and kinetic analysis of thioglycoligase variants. MU release was detected in a fluorescence plate reader.

 
Initial characterization of mutants

Thioglycoligase reactions were carried out with 2.5 mM GlcN3 as donor, 0.7 mM 4SGlcMU as acceptor and 0.25 mg/ml of the appropriate enzyme in 50 mM phosphate buffer pH 7.0. Samples were spotted on TLC plates every 5 min and the amount of product formed was estimated under UV illumination.

Coupled thioglycoligase assay: steady-state kinetic studies

Thioglycoligase reactions were performed using GlcN3 as donor and 4SGlcMU as acceptor with different amounts of acid–base mutants of Abg. Rates were determined by monitoring the release of 4-methylumbelliferone, using EG1 in the coupled assay (Figure 2). Reactions were carried out in the Victor fluorescence plate reader using 96-well plates in 50 mM potassium phosphate buffer pH 7.0 containing 0.1% bovine serum albumin (BSA) and 0.1 mg/ml EG1 at 37°C. The amount of EG1 needed for kinetic analysis such that it was not rate limiting was determined by using a fixed concentration of EG1 (0.1 mg/ml) and different dilutions of thioglycoligase in a series of reactions including 0.28 mM 4SGlcMU and 2 mM GlcN3. This assay showed a linear increase of signal with lower concentrations of thioglycoligase and flattened off at higher concentrations owing to limiting amounts of EG1. Subsequent analyses were carried out such that the observed rate was at least 5-fold below the limit of linearity. To determine the kcat/Km value for the donor sugar, 4SGlcMU (0.28 mM) was used as acceptor and concentrations of GlcN3 were varied from 0.01 to 10 mM. To determine the kcat/Km value for 4SGlcMU, a saturating concentration of GlcN3 (5 mM) was used in combination with various concentrations of acceptor (0.0022–0.28 mM). The emission coefficient (66 450 nmol–1) for 4-methylumbelliferone (MU) was determined by making a dilution curve in 50 mM potassium phosphate, 0.1% BSA and reading 50 µl amounts in the plate reader using the same settings as for the enzyme assays.

Thioglycoligase assay: release of phenolate

Monitoring of the amount of thioglycoligase product formed using dNP-Glc or 4-nitrophenyl ß-D-glucopyranoside (pNP-Glc) as donors, using the coupled assay, was not possible owing to quenching of fluorescence from methylumbelliferone by the nitrophenols released. Reaction progress was therefore monitored by release of the aglycone from the donor sugar. Assays were performed in a Varian spectrophotometer at 37°C using an appropriate concentration of dNP-Glc (0.005–0.1 mM) or pNP-Glc (0.02–1 mM) in the presence of 0.28 mM 4SGlcMU, monitoring the change in absorbance at 405 nm in 50 mM potassium phosphate pH 7.0 and 0.1% BSA. Reactions were pre-equilibrated in the reaction cuvettes at 37°C and started by adding one-tenth the volume of the appropriate enzyme dilution. Extinction coefficients used were 10 900 and 7280 M–1 cm–1 for 2,4-dinitrophenol (dNP) and 4-nitrophenol (pNP), respectively.

For all kinetic analyses, kcat and Km were derived by fitting the obtained data to the Michaelis–Menten equation (Equation 1) or, if necessary, to the equation modified to include substrate inhibition (Equation 2). Non-linear regression analysis was performed using the program GraFit 4.0 (Erithacus Software).

(1)

(2)

Thioglycoligase reactions

A typical small-scale thioglycoligase reaction was performed in 50 mM potassium phosphate pH 7.0 at room temperature. Depending on the mutation, final protein concentrations from 0.001 to 2 mg/ml were used. Acceptor concentrations [either 4-nitrophenyl 4-deoxy-4-thio-ß-D-glucopyranoside (4SGlc-pNP), 4-nitrophenyl N-acetyl-4-deoxy-4-thio-ß-D-glucosaminide (4SGlcNAc-pNP) or 4-nitrophenyl 4-deoxy-4-thio-ß-D-lactopyranoside (4SLac-pNP)] were in the range 0.2–1 mM and donor sugar concentrations ranged from 1 to 5 mM. If not stated otherwise, products were analyzed by TLC and/or electrospray ionization mass spectrometry (ESI-MS). For larger scale reactions, 5–8 mg of acceptor sugar were dissolved in 1 ml of DMF, then twice the molar amount of dNP-Glc or 2,4-dinitrophenyl ß-D-galactopyranoside (dNP-Gal) was added, along with 1 ml of 500 mM potassium phosphate pH 7.0, 8 ml of water and 100 µl of either Abg E170Q (5 mg/ml) or Abg E170G (15 mg/ml). Reactions were left to proceed overnight at room temperature and the products, after lyophilization, standard per-O-acetylation with pyridine–acetic anhydride and subsequent work-up by chromatography on silica gel, were identified by ESI-MS and 1H NMR spectroscopy.

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl)-(1->4)-S-2-(acetamido)-2-deoxy-3,6-di-O-acetyl-4-deoxy-4-thio-ß-D-glucopyranoside: yellowish solid; 1H NMR (400 MHz, {delta} ppm, CDCl3): 8.16 (m, 2 H, aromatic H), 7.08 (m, 2 H, aromatic H), 5.81 (d, 1 H, J1,2 8.0 Hz, H-1), 5.46 (t, 1 H, J2,3 10.0 Hz and J3,4 10.2 Hz, H-3), 5.45 (d, 1 H, J 7.8 Hz, NHAc), 5.20 (t, 1 H, J2',3' 9.1 Hz and J3',4' 9.2 Hz, H-3'), 5.07 (t, 1 H, J4',5' 9.8 Hz, H-4'), 4.92 (t, 1 H, J2',3' 9.0 Hz, H-2'), 4.77 (d, 1 H, J1',2' 10.1 Hz, H-1'), 4.60 (brd, 1 H, J6a',6b' 11.4 Hz, H-6'b), 4.37 (m, 2 H, H-6'a and H-6b), 4.13 (dd, 1 H, J5,6a 4.0 Hz and J6a,6b 12.5 Hz, H-6a), 4.05 (m, 1 H, H-5), 3.98 (dd, 1 H, J1,2 8.0 Hz, H-2), 3.75 (m, 1 H, H-5'), 2.98 (t, 1 H, J4,5 10.6 Hz, H-4), 2.11–1.95 (7s, 21 H, 7 x CH3CO); ESI-MS: m/z = 795 [M + Na]+.

4-Nitrophenyl (2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl)-(1->4)-S-2,3,6-tri-O-acetyl-4-deoxy-4-thio-ß-D-glucopyranoside: white solid; 1H NMR (400 MHz, {delta} ppm, CDCl3): 8.18 (m, 2 H, aromatic H), 7.06 (m, 2 H, aromatic H), 5.43 (d, 1 H, J3',4' 3.1 Hz, H-4'), 5.27–5.20 (m, 2 H, H-3 and H-2), 5.15 (d, 1 H, J1,2 7.5 Hz, H-1), 5.11 (t, 1 H, J1',2' = J2',3' 9.8 Hz, H-2'), 5.02 (t, 1 H, J3',4' 3.3 Hz, H-3'), 4.72 (d, 1 H, J1',2' 9.8 Hz, H-1'), 4.63 (dd, 1 H, J5,6b 1.6 Hz and J6a,6b 12.0 Hz, H-6b), 4.43 (dd, 1 H, J5,6a 5.41 Hz, H-6a), 4.10 (m, 2 H, H-6'a and H-6'b), 4.04(m, 1 H, H-5), 3.93 (m, 1 H, H-5'), 3.03 (t, 1 H, J3,4 10.6 Hz, H-4), 2.16–1.96 (7s, 21 H, 7 x CH3CO); ESI-MS: m/z = 796 [M + K]+.

4-Nitrophenyl [(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl)-(1->4)-S-2,3,6-tri-O-acetyl-4-deoxy-4-thio-ß-D-galactopyranosyl]-(14)-O-2,3,6-tri-O-acetyl-ß-D-glucopyranoside: yellowish solid; 1H NMR (400 MHz, {delta} ppm, CDCl3): 8.18 (m, 2 H, aromatic H), 7.03 (m, 2 H, aromatic H), 5.28 (t, 1 H, J2',3' = J3',4' 9.4Hz, H-3'), 5.23–5.20 (m, 2 H, H-2 and H-3), 5.15 (d, 1 H, J1,2 7.6 Hz, H-1), 5.09 (t, 1 H, J4',5' 9.7 Hz, H-4'), 5.07 (dd, 1 H, J1',2' 7.6 Hz and J2',3' 10.0 Hz, H-2'), 4.95 (dd, 1 H, J3',4' 4.7 Hz, H-3'), 4.90 (dd, 1 H, J1',2' 10.1 Hz, H-2'), 4.66 (d, 1 H, J1',2' 10.1 Hz, H-1'), 4.54 (brd, 1 H, J6a,6b 11.8 Hz, H-6b), 4.37 (d, 1 H, J1',2' 7.7 Hz, H-1'), 4.34–4.31 (m, 2 H, H-6'a and H-6'b), 4.21 (m, 2 H, H-6'a and H-6'b), 4.11 (m, 1 H, H-6a), 3.93 (m, 1 H, H-5'), 3.81 (m, 2H, H-4 and H-5), 3.65 (m, 1H, H-5'), 3.53 (dd, 1 H, J4',5' 1.5 Hz, H-4'), 2.10–1.99 (8s, 30 H, 10 x CH3CO); ESI-MS: m/z = 1084 [M + Na]+.

pH profile of Abg E170Q

Yields of a thioglycoligase reaction at pH values ranging from 5.5 to 9 were determined by reacting 2.5 mM 4SGlcMU and 6.25 mM GlcN3 with 0.06 mg/ml of Abg E170Q (freshly diluted in 0.1% BSA, 100 mM NaCl) in a series of 25 mM buffers containing 100 mM NaCl, 10% DMF. The following buffers were used in an overlapping manner to exclude any effect of the buffer substance on the reaction: MES (pH 5.5–6.5), MOPS (pH 6.5–7.5) and Tris (pH 7–9). After 10 min the reaction was killed in a boiling water-bath for 5 min and 60 µl of 500 mM phosphate buffer pH 7.0 containing 0.02 mg/ml EG1 were added to release MU from the disaccharides formed. After incubation at 37°C for 4 h and room temperature overnight, another 200 µl of 500 mM phosphate buffer were added and the fluorescence of 50 µl aliquots was measured in the Victor plate reader. Relative rates of dNP-Glc hydrolysis by Abg E170Q at different pH values were obtained by monitoring dNP release spectrophotometrically at 405 nm. A saturating concentration (1 mM) of substrate was reacted with Abg E170Q (1.5 mg/ml) at 37°C. Buffers were the same as for the thioglycoligase reaction and it was assumed that the extinction coefficient for dNP (pKa = 3.96) does not change significantly over the observed range. For each series of data, values obtained in Tris buffer at pH 7 were set as 100%.


    Results and discussion
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Screening of the randomized library

Randomization of important residues and subsequent identification of better catalysts have proved to be a successful strategy for improving Abg (Mayer et al., 2001Go) and Humicola insolens Cel7B (Lin et al., 2004Go) glycosynthases. In a similar manner, this paper describes the randomization of the acid–base residue of Abg and screening for variants that are better thioglycoligases than the previously identified Abg E170A. Two different strategies were established to identify active thioglycoligases. The lower throughput approach included monitoring of product formation by TLC, while the higher throughput approach was carried out on plates and employed a coupled assay similar to that used for screening of glycosynthases. In this coupled assay, the acceptor thio sugar used bears a fluorescent leaving group at the anomeric centre (Figure 2). Addition of an endoglycosidase that cleaves the fluorophore only from the extended product allows the identification of active thioglycoligases. Two issues had to be addressed to make the screen work in this context. First, the dNP leaving group of the donor sugar quenched the fluorescence of MU, necessitating the use of an alternative donor. ß-Glucosyl fluoride seemed an ideal candidate, but proved too labile for longer incubations. The donor that proved most useful was GlcN3. Second, the endocellulase Cel5A used in the glycosynthase screen proved to be too specific and would not accept 4-methylumbelliferyl ß-D-glucopyranosyl)-(1 -> 4)-S-4-deoxy-4-thio-ß-D-glucopyranoside (GSGMU) as substrate at useful rates. Fortunately, the endoglucanase from Fusarium oxysporum sp. lycopersi was able to release MU from this compound and hydrolyzed the O-glycosidic linkage next to the S-glycosidic linkage in MU-cellobioside with about 25% of its rate for the parent MU-cellobioside (data not shown). It was also important that neither EG1 nor Abg hydrolyze the acceptor sugar 4SGlcMU. Concentrations up to 0.5 mg/ml of each enzyme were tested with 0.5 mM 4SGlcMU and no hydrolysis was observed. This inactivity of Abg on the bulkier 4-thio sugar substrate is unsurprising given the known specificities of this enzyme (Namchuk and Withers, 1995Go).

After having established the protocol, a library of about 10 000 colonies in which the acid–base catalyst E170 had been randomized by saturation mutagenesis was obtained using a four-primer PCR method. The ligation mixture was transformed, all colonies on the kanamycin plates were collected and plasmid DNA was isolated. To test the quality of the library obtained, six single colonies were picked and sequencing revealed a random distribution of codons at position 170. Two rounds of screening were completed by reacting crude cell lysates of randomly picked colonies with dNP-Glc and 4SGlcMU. The reaction products were analyzed by TLC with product spot detection under UV epi-illumination. In the first test round, 24 clones plus positive (E170A) and negative (E358G) controls were screened: eight of them showed the formation of product and were sequenced. In a second round, 96 colonies, including controls, were screened: 28 of them proved to be active and were sequenced. The mutations found to be active as thioglycoligases at this stage were E170A, E170G, E170N, E170S, E170T, E170F, E170I, E170L, E170M and E170V. The coupled assay was then employed with EG1 as screening enzyme and glucosyl azide as donor (Figure 2), determining the amount of MU released in a fluorescence plate reader. A total of 96 more colonies were screened but surprisingly the only variant found to be active was Abg E170Q. The reasons for this much lower ‘hit rate’ became obvious when thioglycoligase reaction rates with different donor sugars were determined (see below). That Abg E170Q did not show up amongst the positive clones in the TLC screen points towards incomplete coverage of the library at that stage. Although surprising, it is of no significant concern since with both methods together the coverage was sufficient. In total some 220 clones were screened, thus with only 20 amino acids to cover (64 possible codons), full coverage seems almost certain. Indeed, 42 sequences were obtained in positive and negative clones and these encoded 17 of the possible 20 amino acids. Sequences obtained in each case did not contain any unwanted mutations.

Initial characterization of the mutants

The nine mutants of Abg that formed the greatest amount of product in the TLC screen (E170A, E170G, E170N, E170S, E170T, E170F, E170I, E170V and E170L) and E170Q were selected and pure protein preparations thereof were obtained. For initial characterization, protein samples were adjusted to ~5 mg/ml. Each of these was diluted 20-fold and reacted with GlcN3 as donor and 4SGlcMU as acceptor. Aliquots were then taken after every 5 min and analyzed by TLC. For all mutants except E170Q, which had converted all the substrate at the first time point, a time course of product formation was observed. The six mutants with the highest thioglycoligase reaction rates, E170A, E170G, E170N, E170Q, E170S and E170T, were selected for further detailed kinetic characterization.

Kinetic analysis of selected mutants

In order to follow the thioglycoligase reaction rate more directly than by using TLC, a continuous assay to measure the product formation was necessary. Since thioglycoligases, in contrast to glycosynthases, transglycosylate only one sugar residue onto the acceptor the rate of release of MU is directly equivalent to the thioglycoligase rate, making this assay appropriate for enzyme kinetic studies. For this kinetic analysis, thioglycoligase concentrations were chosen such that EG1 was present at concentrations at least 5-fold higher than the limiting value. Michaelis–Menten parameters for the glucosyl azide were determined at a fixed concentration of acceptor sugar 4SGlcMU, this being the highest possible concentration (0.28 mM) achievable. Kinetic parameters determined are presented in Table I. E170Q is a considerably better thioglycoligase than any of the other mutants, with a rate of 3 s–1. Values of kcat for the other acid–base mutants were at least 100-fold lower and ranged from 0.036 s–1 for E170G to 0.003 s–1 for E170T. Km values for GlcN3 were in the range 0.03–0.07 mM for E170G, -S and -T and 0.13–0.59 mM for E170A, -Q and -N. The generally very low Km values observed for GlcN3 are all consistent with relatively rapid formation of a glycosyl enzyme intermediate and rate-limiting breakdown via transglycosylation (Wang et al., 1995Go). Interestingly, the highest Km values were measured for the amide mutants, implying some steric hindrance to binding or, more likely, that the amide is providing some assistance to the deglycosylation step, possibly via hydrogen bonding with the incoming thio sugar acceptor. Kinetic parameters for 4SGlcMU were determined similarly, using a single fixed (5 mM) concentration of GlcN3. Substrate inhibition was observed for all mutants, with Ki values in the range from 0.4 mM for E170G to 1.1 mM for E170N (Table I). Since, as discussed earlier, 4-thioglucose cannot occupy the –1 subsite of Abg, this substrate inhibition is most likely due to unproductive binding of the acceptor in the +1 subsite, thereby excluding the donor sugar from the active site. Kinetic data obtained for E170S and fitted to the equation for substrate inhibition (Equation 2) are shown in Figure 3. The generally higher rates that were observed when using the donor at a fixed concentration are exactly as expected since GlcN3 was present at truly saturating levels, whereas this could not be the case for the other set of data. Km values for 4SGlcMU were generally in the range 0.03–0.06 mM, except for E170Q with a value of 0.29 mM. The higher value in this case may be due to steric hindrance, since Q is the largest substituent and sulfur is larger than natural oxygen. Comparison of reaction efficiencies (kcat/Km) for donor and acceptor sugar with different mutants (Figure 4) showed clearly the superiority of Abg E170Q compared with any other mutant isolated. Noticeably, in contrast to the other mutants, Abg E170N shows a relatively low kcat/Km for GlcN3 compared with its kcat/Km for 4SGlcMU. In that case, as mentioned earlier, it seems that the glycosylation step (as reflected in kcat/Km) is for some reason relatively less efficient for that mutant than for the others. However, more detailed kinetic analysis is necessary to understand this better.


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Table I. Kinetic parameters for thioglycoligase variants

 


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Fig. 3. Kinetic data for the thioglycoligase reaction carried out with Abg E170S fitted to Equation 2 for substrate inhibition. GlcN3 was kept at a fixed concentration while 4SGlcMU was varied. The inset shows the Lineweaver–Burk representation of the same set of data.

 


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Fig. 4. Catalytic efficiency of thioglycoligase variants for GlcN3 as donor and 4SGlcMU as acceptor. Note that the axis for kcat/Km is split to accommodate a wider range.

 
Thioglycoligase reactions with different donor sugars

For a direct comparison of the utility of different donor sugars for thioglycoligase reactions, kinetic parameters were determined for Abg E170Q and E170A with dNP-Glc and pNP-Glc in the presence of 0.28 mM 4SGlcMU as acceptor and the results were compared with those for GlcN3 (Table II). In these cases, the reaction could be directly monitored spectrophotometrically via phenolate release. As judged from analysis of TLCs, reactions in the presence of acceptor thio sugar proceed via transglycosylation and not hydrolysis. The best donor substrate by far, not surprisingly, is dNP-Glc with kcat/Km values approximating those measured for the wild-type enzyme. Hence the excellent dNP leaving group is essentially completely chemically complementing the absence of the acid catalyst in the first step. The consequence is a very low donor Km value, as seen previously (Wang et al., 1995Go). pNP-Glc and GlcN3 are comparable in their kinetic behavior with kcat/Km values four orders of magnitude lower than for dNP-Glc, consistent with the differences in leaving group abilities, at least of the phenols (pKa for dNP = 3.96 and for pNP = 7.18). Despite the fact that azide has a low pKa of 4.72 it is not as good a leaving group as a comparable oxygen-containing leaving group because of the greater inherent strength of the C–N bond than the C–O bond. Furthermore, the phenolate leaving group might provide some transition state stabilization that the azide cannot provide. Therefore, direct comparisons of rates cannot be made. Importantly, however, E170Q is ~100-fold better in each case than E170A, based on kcat/Km values, suggesting that the amide side chain in this enzyme is able to assist the departure of the poorer leaving groups. These results nicely explain the greater stringency established by the coupled assay in screening of mutants. Even though the mutants were capable of product formation from the GlcN3 donor, the amounts formed were too low for reliable detection.


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Table II. Kinetic parameters for thioglycoligases with different donor sugars

 
pH profile of transglycosylation by AbgE170Q

pH profiles of wild-type glycosidases usually show an acidic and basic limb that can be attributed to ionization of the nucleophile and acid–base catalyst, respectively. In earlier work, pH profiles of Abg and Abg E170G were analyzed (Kempton and Withers, 1992Go; Wang et al., 1995Go) and pKa values of <5 and 8.0 thereby assigned to the nucleophile and acid–base catalyst, respectively. No pH dependence was observed for E170G, in the range tested, consistent with the removal of the acid–base catalyst, along with possible displacement of the pKa of the nearby nucleophile out of the observable range. The pH profile obtained in the current study (Figure 5, closed symbols) shows very clearly that the efficiency of the thioglycoligase reaction drops significantly at pH values <7. Consistent with the data for E170G, hydrolysis of dNP-Glc by E170Q (open symbols) does not show any pH dependence. The simplest interpretation of this difference in pH dependence is that the pKa observed in Figure 5 is that of the sugar thiol bound to the enzyme, with the active species being the thiolate anion. The lower apparent pKa observed here (≤6) relative to that of a normal alkanethiol (9–10) is presumably a consequence of the enzymatic environment in this mutant, which might be expected to provide some stabilization of an anion given that an anion is normally found at this site. It is also consistent with earlier observations that wild-type Abg does not utilize thio sugar acceptors (Jahn et al., 2003Go).



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Fig. 5. Logarithmic plot of relative activities versus pH of thioglycoligase and hydrolysis reactions for Abg E170Q. The yields of the thioglycoligase reaction for Abg E170Q increase very slightly from pH 7 upwards but drop drastically below. Hydrolysis of dNP-Glc by Abg E170Q is independent of pH over the observed range. The rate for the thioglycoligase reaction using GlcN3 as donor is 20-fold faster than for hydrolysis of dNP-Glc at pH 7. Closed symbols are for the thioglycoligase reaction and open symbols for hydrolysis. Three buffers [MES (circle), MOPS (square) and Tris (triangle)] were used in an overlapping manner to exclude any buffer effect.

 
Synthesis of thio-linked di- and trisaccharides

Having characterized the most active thioglycoligases, some insights into the true synthetic utility of these enzymes with other sugar pairs was desirable. Of special interest were the acceptor sugars pNP-4SGlcNAc and pNP-4SLac, and, since Abg is also a very effective ß-galactosidase, with dNP-Gal as donor. Thioglycoligase reactions were carried out with both E170G and E170Q to ensure that the reaction was working, then scaled up to the 5–8 mg scale. Results are summarized in Table III. NMR analysis (see Materials and methods) confirmed the product structure in each case. Much to our surprise, the mutant that was the most efficient at transferring glucose units onto 4-thioglucosides (E170Q) was not necessarily the best mutant for other reactions or, as was the case for galactosyl transfer, did not work at all. Indeed, for both transfer of galactose or for transfer of other sugars onto 4-thiogalactose, the E170G enzyme was the most effective. The reasons for this behavior are not clear and would require further analysis. However, it appears that transfer of glucose to the 4-position of 4-thioglucosides is optimally accommodated within the active site, with the E170Q amide side chain providing some assistance to catalysis. In contrast, perhaps the axial thiols on the lactose acceptor require a more flexible active site, with the less bulky glycine at this position permitting such flexibility.


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Table III. Thioligase reaction products and yields

 
Conclusions

A variety of acid–base mutants of Abg were created and several were selected that function as thioglycoligases. In order to do so, the screen developed for the directed evolution of the Abg glycosynthase was modified by introducing glycosyl azides as donor sugars for thioglycoligase reactions and using EG1 as the coupling enzyme. This screening setup also proved useful for kinetic analysis of the isolated mutants. The resultant Abg thioglycoligases proved useful for chemo-enzymatic synthesis of a variety of thio-linked saccharides, with a range of efficiencies. The underlying reasons for these different rates are unclear and require a more detailed kinetic analysis. Hopefully this will result in a better understanding of the thioglycoligase reaction and will be helpful in the design of thioglycoligases of this and other glycosidases.


    Acknowledgments
 
We thank the Protein Engineering Network of Centres of Excellence and the Natural Sciences and Engineering Research Council of Canada for financial support. J.M. was financed by a Schrödinger fellowship from the Austrian FWF. We are grateful to Emily Kwan for technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received November 18, 2004; revised January 19, 2005; accepted January 20, 2005.

Edited by Dan Tawfik





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