Glycobiology Advance Access originally published online on February 9, 2008
Glycobiology 2008 18(4):325-330; doi:10.1093/glycob/cwn011
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase
2 Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa 921-8836
3 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657
4 National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
1 To whom correspondence should be addressed: Tel: +81-29-838-8071; Fax: +81-29-838-7321; e-mail: mkitaoka{at}affrc.go.jp
Received on October 3, 2007; revised on December 26, 2007; accepted on February 3, 2008
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F– releasing activity from
-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F– releasing activity. As a result of the high F– releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact. Key words: general base / glycosyl fluoride / glycosynthase / inverting glycoside hydrolase / reducing-end xylose-releasing exo-oligoxylanase
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Glycoside hydrolases (GHs) are generally categorized into two types, retaining and inverting enzymes, on the basis of changes in their anomeric configurations during hydrolytic reactions (Sinnott 1990
; Henrissat 1991; Henrissat and Bairoch 1993, 1996; Davies and Henrissat 1995). Both these enzymes have two acidic catalytic residues that act as a general acid (proton donor) and a general base (nucleophile). The mechanism of a typical retaining GH is a double displacement reaction, in which the nucleophile directly attacks the anomeric center of the glycoside with the general acid donating a proton to the glycosyl oxygen atom, producing a covalently bound intermediate of the nucleophile with a Walden inversion, followed by hydrolysis of the intermediate with another Walden inversion. In contrast, the mechanism of a typical inverting GH is a single displacement reaction, in which a water molecule activated by the catalytic base attacks the anomeric center to hydrolyze the glycoside with the aid of the general acid, resulting in an anomeric inversion. The transglycosylation activity is often used to prepare oligosaccharides by retaining GHs. However, inverting GHs theoretically do not possess the transglycosylation activity because the reaction of inverting GHs is irreversible once the linkage is cleaved with the addition of a water molecule, which is abundant in the reaction medium. Thus, the use of inverting GHs has been theoretically limited to hydrolysis and not synthesis reactions.
Glycosynthase is a nucleophile-mutant retaining GH that catalyzes the synthesis of glycosides from the glycosyl fluoride of the opposite anomer. It was first reported by Mackenzie et al. (1998) on a retaining exo-β-glycosidase, β-glucosidase from Agrobacterium sp., with a mutation in its nucleophilic residue (E358). Then, glycosynthases from endo-β-glycosidase (1,3-1,4-β-glucanase from Bacillus licheniformis) (Malet and Planas 1998) and exo--glycosidase (-glucosidase from Schizosaccharomyces pombe) (Okuyama et al. 2002) followed. To date, various retaining GHs have been converted into glycosynthases through substitutions in their nucleophilic residues (Hancock et al. 2006; Faijes and Planas 2007).
We have attempted to convert an inverting hydrolase, the reducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.15
[EC]
6) belonging to GH family 8 into glycosynthase. Rex is a strict reducing-end-specific exo-lytic enzyme liberating the xylose residue at the reducing end of xylooligosaccharide whose degree of polymerization is 3 or higher (Honda and Kitaoka 2004; Fushinobu et al. 2005) (Figure 1A). The wild-type Rex exhibited the Hehre resynthesis-hydrolysis mechanism (Hehre et al. 1979; Williams and Withers 2000) in which -xylobiosyl fluoride (-X2F) was hydrolyzed to xylobiose (X2) and HF only in the presence of xylose (X1) as an acceptor molecule via the formation of xylotriose (X3) as an undetectable intermediate (Honda and Kitaoka 2006) (Figure 1B). Then, the saturation mutagenesis was performed at the general base residue D263 of Rex to weaken the hydrolysis step of the Hehre resynthesis-hydrolysis mechanism. We found that the D263C and D263N mutants of Rex accumulated significant amounts of X3 from -X2F and X1, indicating that Rex was converted into glycosynthases (Honda and Kitaoka 2006). This finding expanded the glycosynthase world to inverting GHs, making it possible to use them in synthesis reactions.
|
3) to form β-anomer of X1 and However, the Rex glycosynthase retained the significant hydrolytic activity that decreased the yield of the synthetic product. Moreover, the F– releasing activities of the mutants were much lower than that of the wild type (Honda and Kitaoka 2006
). The mutation at the general base residue of an inverting GH could not remove the hydrolytic activity completely because the water molecule retained some activity as a nucleophilic reagent without the aid of the residue (Honda and Kitaoka 2006). It is therefore still important to reduce the hydrolytic activity of glycosynthase obtained from inverting GHs.
Collins et al. (2002, 2005) found that a mutation at a conserved tyrosine residue (Y203) in GH-8 endo-xylanase from Pseudoalteromonas haloplanktis (pXyl), which shares 32.6% amino acid sequence identity with Rex, causes a drastic decrease in the hydrolytic activity of the enzyme toward xylan. The residue formed a hydrogen bond with the nucleophilic water molecule that had another hydrogen bond with the general base residue (D281). The pH dependence of the Y203F mutant indicated that the tyrosine residue was not a general base, but was important to locate the nucleophilic water at the proper position (Collins et al. 2005).
We have found Rex to be a suitable tool to examine glycosynthase. As Rex hydrolyzes X3 to release X1 from the reducing end, it is expected to utilize -X2F as the donor and X1 as the acceptor. It should be mentioned that the hydrolytic product of -X2F, X2 will never act as the acceptor molecule because of the subsite structure of Rex (Fushinobu et al. 2005) (Figure 1). Thus, the donor and the acceptor are completely independent during the reaction (Honda and Kitaoka 2006). The idea led us to continue glycosynthase research on Rex. In this study, we describe an alternative design of a glycosynthase from Rex in which we mutated the corresponding tyrosine residue, which is not the general base residue.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Determination of the tyrosine residue in Rex
A water molecule was found to form a hydrogen bond with the catalytic base residue D263 in the structure of Rex (PDB accession no. 1WU4) (Fushinobu et al. 2005
, Honda et al. 2005). It formed another hydrogen bond with the phenolic oxygen atom of Y198 (Figure 2). Because this is the only water molecule that directly interacts with D263, it was considered as a nucleophile in the hydrolysis. Y198 of Rex was found to correspond to Y203 of pXyl (Collins et al. 2005) in both sequence alignment (data not shown) and structural comparison (Figure 2). Then, Y198 of Rex was replaced by phenylalanine with and without the displacement of the general base residue (D263).
|
Activity of Y198F mutants
The F– releasing activity and hydrolytic activities of the mutants at Y198 and/or D263 are summarized in Table I. The single mutation at Y198 drastically decreased the hydrolytic activity (from 31.2 to 0.06 s–1), with a small increase in the F– releasing activity. The F– releasing activity of Y198F was more than 10 times that of the mutants at the general base residue (D263). The ratio of F– releasing activity by the hydrolytic activity (F/H) was 78, approximately four times that of the best glycosynthase mutant at the general base residue (D263C). Double mutation was also tested. Y198F/D263N showed 20 times less F– releasing activity than Y198F, which was comparable to that of the corresponding single mutant, D263N. The F/H ratio (144) of D263N was two times higher than that of Y198F. The Y198F/D263C mutant showed only faint F– releasing activity. The wild type, Y198F, and Y198F/D263N showed no activity on
-X2F in the absence of X1, even though significant amounts of -X2F were consumed in the presence of X1 under the same conditions (Figure 3).
|
|
Kinetic analyses of the glycosynthase reaction
The apparent kinetic parameters of the glycosynthase reaction on
-X2F and X1 in the presence of 10 mM X1 and -X2F, respectively, are summarized in Table II. The kcat and Km values of Y198F and Y198F/D263N as well as that of the wild type on -X2F were not calculated because their Sv curves were linear up to 20 mM. The kcat/Km of Y198F was 1.5 times that of the wild type. On the other hand, the kcat/Km of Y198F/D263N was 1/10 times that of the wild type. The kcat and Km values of Y198F on X1 were approximately double that of the wild type, resulting in a similar kcat/Km. For Y198F/D263N, the kcat was 1/10 times that of the wild type, with a similar Km value.
|
The kinetic parameters of X3 hydrolytic activity by Y198F were also determined. The Km value of Y198F (4.3 mM) was similar to that of wild type (6.4 mM), whereas its kcat value (0.22 s–1) was 0.27% that of wild type (83 s–1).
Time course of the glycosynthase reaction by Y198F and Y198F/D263N
The reaction products from -X2F and X1 produced by Y198F and Y198F/D263N gave the 1H- and 13C-NMR spectra identical with those of authentic X3 (see Supplementary data). Time courses of the reactions with 5 mM -X2F and X1 by Y198F and Y198F/D263N were compared with that catalyzed by the best base mutant D263C (Figure 4). Much greater accumulation of X3 (glycosynthase product) and much less formation of X2 (hydrolytic product from X3) were observed in the reactions catalyzed by Y198F and Y198F/D263N than in that catalyzed by D263C. The ratio of X3 in the products {X3/(X2 + X3)} when half of -X2F was consumed was 0.59 (D263C), 0.93 (Y198F), and 0.96 (Y198F/D263N). Although Y198F/D263N showed a slightly larger X3 ratio than Y198F, the F– releasing activity of Y198F was 20 times that of Y198F/D263N. Note that the experiments shown in Figure 2B and C were performed with 0.1 µM Y198F and 7.7 µM Y198F/D263N, respectively. These results suggest that Y198F was the best glycosynthase of the mutants.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Glycosynthase is useful for converting GHs into a catalyst for sugar chain synthesis (Mackenzie et al. 1998
). To date, all glycosynthases derived from retaining GHs have been generated by mutating their nucleophilic acidic residues (Malet and Planas 1998; Okuyama et al. 2002; Trincone et al. 2005; Faure et al. 2006; Hancock et al. 2006; Kim et al. 2006; Mullegger et al. 2006; Sugimura et al. 2006; Faijes and Planas 2007; Hommalai et al. 2007). Prior to this report, the glycosynthase of Rex, the only glycosynthase derived from an inverting GH, was also created by mutating the general base residue D263 (Honda and Kitaoka 2006). However, our observations showed that the wild-type Rex exhibited the highest F– releasing activity among the mutants at the nucleophilic residue and concluded that it was important to minimize the decrease in the F– releasing activity while maximizing the decrease in the hydrolytic activity to generate a glycosynthase from an inverting GH. Thus, we proposed that a mutation at a residue other than the general base D263 that the decreased hydrolytic activity would generate an effective glycosynthase of Rex.
Collins et al. (2005) reported that the Y203F mutant of pXyl has drastically decreased the hydrolytic activity toward xylan. The tyrosine residue holds a nucleophilic water molecule with the general base aspartic acid residue. Mutation of the tyrosine residue in Rex (Y198) into phenylalanine resulted in 1/500 of the hydrolytic activity toward X3 without a decrease in the F– releasing activity as expected. de vos D et al. (2006) also pointed out that the residue in pXyl might be important for binding the xylan chain in the correct direction by forming an indirect hydrogen bond through another water molecule to the endocyclic O5 in the xylosyl residue binding at subsite –2. Such a role is negligible for Rex because the mutation at Y198 did not significantly change the Km value toward X3 in the hydrolytic reaction and the kcat/Km value toward -X2F in the glycosynthase reaction.
The double mutant, Y198F/D263N, had a better F/H ratio than did Y198F; however, its F– releasing activity was much less than that of Y198F. This result suggests that the general base residue should not be substituted in order to obtain high F– releasing activity. We propose a novel concept for creating a glycosynthase from an inverting GH by mutating a residue holding the nucleophilic water molecule with the general base residue while keeping the general base residue intact.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Reagents
Xylooligosaccharides (Xn, where n = degree of polymerization) were purchased from Megazyme (Wicklow, Ireland).
-X2F was synthesized by reacting hexa-O-acetyl xylobiose with pyridinium poly(hydrogen fluoride), followed by O-deacetylation with sodium methoxide in methanol according to the standard procedure (Hayashi et al. 1984; Yokoyama 2000). Other analytical grade reagents were obtained from various commercial sources.
Construction of Rex mutants
Genes encoding wild type, D263N, and D263C Rex from Bacillus halodurans C-125 containing the His x 6 sequence at the C-terminal end were constructed as reported previously (Honda and Kitaoka 2004). To construct Y198F, D263N/Y198F, and D263C/Y198F Rex, site-directed mutagenesis was performed by PCR with a KOD polymerase (Toyobo, Osaka, Japan). In the reaction, pET28b plasmids of wild type, D263N, and D263C were used as the templates. The following mutagenic primers were used (the mismatched base is underlined): 5'-AGT GAT CCC TCT TTT CAT CTC CCC CAT TTT-3' (forward primer) and 5'-G GAG ATG AAA AGA GGG ATC ACT GAA CTC CA-3' (reverse primer). The reaction solution was digested with DpnI. Next, pET28b plasmids for Y198F, D263N/Y198F, and D263C/Y198F were transformed into Escherichia coli BL21 (DE3) cells, and positive colonies were selected.
Preparation of Rex mutants
Each transformant was incubated in Luria broth (100 mL) containing 0.05 mg/mL kanamycin at 37°C until the optical density, at 600 nm, reached 0.6. Isopropyl-β-D-thiogalactopyranoside was then added to give a final concentration of 1 mM, and the cultures were incubated for 22 h at 25°C. The expressed Rex mutant proteins were extracted from the wet cells using Bug Buster(TM) HT (Novagen, Darmstadt, Germany). The cell-free extract was loaded onto a Ni-NTA agarose (Qiagen, Hilden, Germany) column (1 x 3 cm), and the enzyme was eluted with a stepwise gradient of imidazole (1, 10 mM; 2, 20 mM; 3, 250 mM) in a 50 mM sodium phosphate buffer (pH 8.0) containing 0.3 M NaCl.
Finally, Y198F, D263N/Y198F, and D263C/Y198F were desalted using an Amicon Ultra-4-10k (Millipore,Carrigtwohill, Ireland). The purity of the proteins was checked by SDS–PAGE (Laemmli 1970). Protein concentrations were determined by measuring absorbance at 280 nm based on the theoretical molar absorption coefficients (106,210 M–1 cm–1) as described previously (Pace et al. 1995).
Enzyme assay
The enzymatic F– release reaction was performed in a 0.1 M MOPS buffer (pH 7.0) containing 10 mM -X2F and X1 at 30°C. The reaction was monitored using an F–-selective electrode (9609 BN, Thermo Orion, Beverly, MA) interfaced with a portable pH/ISE Meter model 290A+ (Thermo Orion). A F– standard curve was determined using sodium fluoride solution under the same reaction conditions without enzymes and substrates. The rate constant of the spontaneous hydrolysis of -X2F was determined to be 2.6 x 10–6 s–1 and the half-life of -X2F was 2.7 x 105 s under the above conditions. All the glycosynthase experiments in this report were designed to make the spontaneous hydrolysis of -X2F negligible.
To determine the apparent kinetic parameters of the glycosynthase reaction, -X2F and X1 were subjected to F– release in a 0.1 M MOPS buffer (pH 7.0) at 30°C. The initial rates were measured as fluoride ions increased, as described above. The kinetic parameters were calculated by regressing the experimental data (substrate concentration range: 0.2–3 Km) with the Michaelis–Menten equation by the curve-fitting method using Kaleidagraph(TM) ver. 4.00 (Synergy Software, Reading, PA).
To determine the apparent kinetic parameters of X3 hydrolytic activity, X3 was subjected to hydrolysis in a 0.1 M MOPS buffer (pH 7.0) at 30°C. The initial rates were determined by measurements of the increase in xylose by high-performance ion-exchange chromatography (HPIC), as described above. The kinetic parameters were calculated by regressing the experimental data (substrate concentration range: 0.2–3 Km) with the Michaelis–Menten equation by the curve-fitting method.
Analysis of the reaction products
The reaction products from -X2F and X1 were separated by thin-layer chromatography on silica gel 60 F254 plates (5.0 x 7.5 cm; Merck, Darmstadt, Germany) with a solvent system of acetonitrile/water (4:1, v/v). Sugars were detected by baking after dipping the plates in 5% sulfuric acid in methanol. When necessary, the amounts of the products were quantified by HPIC on a CarboPac(TM) PA1 column (2 x 250 mm, Dionex, Sunnyvale, CA) equipped with a pulsed amperometric detector (ICS-3000, Dionex). Chromatography was performed with a linear gradient of 0–0.2 M sodium acetate in 0.1 M NaOH for 20 min at a flow rate of 0.25 mL/min.
Structural analysis of the product
-X2F (2.8 mg, 10 µmol) and X1 (1.5 mg, 10 µmol) were dissolved in 0.5 mL of 0.1 M MOPS buffer (pH 7.0) and mixed with Y198F (2.7 nmol) or Y198F/D263N (5.9 nmol). The reaction mixture was incubated for 2 h at 30°C, followed by deionization with Amberlite MB-3 (Organo, Tokyo, Japan), and lyophilized. The products were isolated by HPLC using a TSK-GEL Amido-80 column (4.6 x 250 mm, Tosoh, Japan) and eluted with acetonitrile/water (7:3 v/v) at a flow rate of 1.5 mL/ min at 25°C. The products were detected with a refractive index monitor (RID 10A, Shimadzu, Kyoto, Japan). The fractions containing the products were evaporated and lyophilized to obtain white powders (Y198F: yield, 1.1 mg, 2.7 µmol, 27%; Y198F/D263N: yield, 1.3 mg, 3.1 µmol, 31%). The 1H-NMR and 13C-NMR spectra of the products were taken in D2O using the Bruker Avance800 spectrometer and Bruker Avance500 spectrometer, respectively, and were compared with those of authentic X3.
Time course of the glycosnthase reaction
The enzymatic reactions by D263C, Y198F, and D263N/Y198F were performed in a 0.1 M MOPS buffer (pH 7.0) containing 5.0 mM -X2F and X1 at 30°C. At appropriate reaction times, aliquots of the reaction solution were withdrawn and diluted with distilled water (1:100). The solutions were subjected immediately to HPIC and analyzed as described above. In the enzymatic reaction, fluoride ions in the reaction solution were also detected using a fluoride electrode as described above.
|
Supplementary Data |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
|
Funding |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Ministry of Education, Culture, Sports, Science, and Technology, Japan (17780086 to Y.H.; and 19380062 to Y.H. and H.T.), Research Fellowships of the Japan Society for the Promotion of Science for Young Scientist (17-182 to M.H.), and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
None declared.
|
Abbreviations |
|---|
GH, glycoside hydrolase; HPIC, high-performance ion-exchange chromatography; MOPS, 3-morpholinopropane- sulfonic acid; pXyl, GH8 endo-xylanase from Pseudoalteromonas haloplanktis; Rex, reducing-end xylose-releasing exo-oligoxylanase; Xn, xylooligosaccharide with n degrees of polymerization; X1, xylose;
-X2F, -xylobiosyl fluoride |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
|---|
Collins T, De Vos D, Hoyoux A, Savvides SN, Gerday C, Van Beeumen J, Feller G. Study of the active site residues of a glycoside hydrolase family 8 xylanase. J Mol Biol (2005) 354:425–435.[CrossRef][Web of Science][Medline]
Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C. A novel family 8 xylanase, functional and physicochemical characterization. J Biol Chem (2002) 277:35133–35139.
Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure (1995) 3:853–859.[Medline]
de vos D, Collins T, Nerinckx W, Savvides SN, Claeyssens M, Gerday C, Feller G, Van Beeumen J. Oligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the β-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product. Biochemistry (2006) 45:4797–4807.[CrossRef][Web of Science][Medline]
Faijes M, Planas A. In vitro synthesis of artificial polysaccharides by glycosidases and glycosynthases. Carbohydr Res (2007) 342:1581–1594.[CrossRef][Web of Science][Medline]
Faure R, Saura-Valls M, Brumer H 3rd, Planas A, Cottaz S, Driguez H. Synthesis of a library of xylogluco-oligosaccharides for active-site mapping of xyloglucan endo-transglycosylase. J Org Chem (2006) 71:5151–5161.[CrossRef][Web of Science][Medline]
Fushinobu S, Hidaka M, Honda Y, Wakagi T, Shoun H, Kitaoka M. Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125. J Biol Chem (2005) 280:17180–17186.
Hancock SM, Vaughan MD, Withers SG. Engineering of glycosidases and glycosyltransferases. Curr Opin Chem Biol (2006) 10:509–519.[CrossRef][Web of Science][Medline]
Hayashi M, Hashimoto M, Noyori R. Simple synthesis of glycosyl fluorides. Chem Lett (1984) 1747–1750.
Hehre EJ, Brewer CF, Genghof DS. Scope and mechanism of carbohydrase action. Hydrolytic and nonhydrolytic actions of beta-amylase on alpha- and beta-maltosyl fluoride. J Biol Chem (1979) 254:5942–5950.
Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J (1991) 280:309–316.[Web of Science][Medline]
Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J (1993) 293:781–788.[Web of Science][Medline]
Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochem J (1996) 316:695–696.[Web of Science][Medline]
Hommalai G, Withers SG, Chuenchor W, Cairns JR, Svasti J. Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 β-glucosidase glycosynthase mutants. Glycobiology (2007) 17:744–753.
Honda Y, Fushinobu S, Hidaka M, Wakagi T, Shoun H, Kitaoka M. Crystallization and preliminary X-ray analysis of reducing-end-xylose releasing exo-oligoxylanase from Bacillus halodurans C-125. Acta Cryst F (2005) 61:291–292.[CrossRef]
Honda Y, Kitaoka M. A family 8 glycoside hydrolase from Bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase. J Biol Chem (2004) 279:55097–55103.
Honda Y, Kitaoka M. The first glycosynthase derived from an inverting glycoside hydrolase. J Biol Chem (2006) 281:1426–1431.
Kim YW, Fox DT, Hekmat O, Kantner T, McIntosh LP, Warren RA, Withers SG. Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi. Org Biomol Chem (2006) 4:2025–2032.[CrossRef][Web of Science][Medline]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
Mackenzie LF, Wang Q, Warren RAJ, Withers SG. Glycosynthases: Mutant glycosidases for oligosaccharide synthesis. J Am Chem Soc (1998) 120:5583–5584.[CrossRef][Web of Science]
Malet C, Planas A. From beta-glucanase to beta-glucansynthase: Glycosyl transfer to alpha-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile. FEBS Lett (1998) 440:208–212.[CrossRef][Web of Science][Medline]
Mullegger J, Chen HM, Warren RA, Withers SG. Glycosylation of a neoglycoprotein by using glycosynthase and thioglycoligase approaches: The generation of a thioglycoprotein. Angew Chem Int Ed Engl (2006) 45:2585–2588.[CrossRef]
Okuyama M, Mori H, Watanabe K, Kimura A, Chiba S. Alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction. Biosci Biotechnol Biochem (2002) 66:928–933.[CrossRef][Medline]
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci (1995) 4:2411–2423.[Web of Science][Medline]
Sinnott ML. Catalytic mechanism of enzymic glycosyl transfer. Chem Rev (1990) 90:1171–1202.[CrossRef][Web of Science]
Sugimura M, Nishimoto M, Kitaoka M. Characterization of glycosynthase mutants derived from glycoside hydrolase family 10 xylanases. Biosci Biotechnol Biochem (2006) 70:1210–1217.[CrossRef][Medline]
Trincone A, Giordano A, Perugino G, Rossi M, Moracci M. Highly productive autocondensation and transglycosylation reactions with Sulfolobus solfataricus glycosynthase. Chembiochem (2005) 6:1431–1437.[CrossRef][Web of Science][Medline]
Williams SJ, Withers SG. Glycosyl fluorides in enzymatic reactions. Carbohydr Res (2000) 327:27–46.[CrossRef][Web of Science][Medline]
Yokoyama M. Methods of synthesis of glycosyl fluorides. Carbohydr Res (2000) 327:5–14.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||