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University of Groningen Engineering the Glucansucrase GTFR University of Groningen Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity Hellmuth, Hendrik; Wittrock, Sabine; Kralj, Slavko; Dijkhuizen, Lubbert; Hofer, Bernd; Seibel, Juergen; Seibel, Jürgen Published in: Biochemistry DOI: 10.1021/bi800563r IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hellmuth, H., Wittrock, S., Kralj, S., Dijkhuizen, L., Hofer, B., Seibel, J., & Seibel, J. (2008). Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: Toward tailor-made polymer and oligosaccharide products. Biochemistry, 47(25), 6678-6684. https://doi.org/10.1021/bi800563r Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 01-10-2021 6678 Biochemistry 2008, 47, 6678–6684 Engineering the Glucansucrase GTFR Enzyme Reaction and Glycosidic Bond Specificity: Toward Tailor-Made Polymer and Oligosaccharide Products† Hendrik Hellmuth,‡ Sabine Wittrock,§ Slavko Kralj,| Lubbert Dijkhuizen,| Bernd Hofer,§ and Ju¨rgen Seibel*,‡,⊥ Department of Carbohydrate Technology, UniVersity of Braunschweig, Braunschweig, Germany, DiVision of Structural Biology and Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany, and Centre for Carbohydrate Bioprocessing, TNO-UniVersity of Groningen, and Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, UniVersity of Groningen, Groningen, The Netherlands ReceiVed April 1, 2008; ReVised Manuscript ReceiVed May 6, 2008 ABSTRACT: Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly R-1,6-glucosidic linkages to a mutan type polymer with predominantly R-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency. The synthesis of oligosaccharides and polysaccharides is promising variations in structural and functional properties, a challenging goal for modern science. Despite the great which needs to be elucidated further. Known are dextrans importance and the enormous potential of oligosaccharides, with different structural specificities [R-1,6-bound glucose their technical and pharmaceutical usage is still limited (Glc) backbone, with R-1,2 and R-1,3 side chains] (4, 5), because of the high complexity of these molecules, causing mutan (R-1,3-bound Glc units) (6), alternan (R-1,6- and enormous problems in classical chemical synthesis (1). The R-1,3-bound Glc units) (7), and amylose/reuteran (R-1,4- production of tailor-made oligosaccharides and polysaccha- bound Glc units) (8). In addition to defined polymers, ride structures is still a growing field of interest in modern oligosaccharides with specific structures are urgently needed. biotechnology. An established industrial process is the Oligosaccharides currently produced for commercial markets fermentative production of polysaccharides (R-glucans) by (9), including isomaltooligosaccharides (IMO)1 (10), leucrose lactic acid bacteria, e.g., dextran by Leuconostoc mesenteroi- (11), and palatinose (12), are of interest in the fields of food, des. Such polymers are used in the food industry, as additives pharmacy, and cosmetics because of their ability to prevent for dyes, and in health care (2, 3). Differences in the and treat diseases from various biological origins. Isomaltose, glycosidic linkage type, the degree and type of branching, for instance, enhances cytokine IL-12 production by mac- and the molecular mass of glucans available at present show rophages stimulated with Lactobacillus gasseri in vitro, and dietary IMO significantly increased the number of lactobacilli in the intestinal microflora (13). For the synthesis of † This work was supported by the German Research Foundation via oligosaccharides, the concept of so-called glycosynthases is Sonderforschungsbereich 578 “From Gene to Product” (to H.H., S.W., B.H., and J.S.) and by the EET program of the Dutch government very promising, where the hydrolytic activity of glycosidases (Project EETK01129) (to S.K. and L.D.). has been suppressed via random and rational site-directed * To whom correspondence should be addressed: Division of mutagenesis, allowing transglycosylation reactions to occur Structural Biology, Helmholtz Centre for Infection Research, Inhof- fenstrasse 7, D-38124 Braunschweig, Germany. Telephone: ++49531/ instead (14–18). Here we report an alternative approach that 6181-7001. Fax: ++49531/6181-7098. E-mail: juergen.seibel@ switches the regioselectivity and acceptor specificity of helmholtz-hzi.de. GTFR of Streptococcus oralis, a dextran-producing enzyme, ‡ University of Braunschweig. by random mutagenesis toward synthesis of (a) various short § Department of Chemical Biology, Helmholtz Centre for Infection Research. | University of Groningen. 1 Abbreviations: GTFR, glycosyltransferase R; IMO, isomaltooli- ⊥ Division of Structural Biology, Helmholtz Centre for Infection gosaccharide; HPAEC, high-performance anion exchange chromatog- Research. raphy; PCR, polymerase chain reaction. 10.1021/bi800563r CCC: $40.75 2008 American Chemical Society Published on Web 05/31/2008 Glucansucrase GTFR Reaction and Glycosidic Bond Specificity Biochemistry, Vol. 47, No. 25, 2008 6679 chain oligosaccharides or (b) novel (mutan) polymers with GTFR Library Screen for Enzymatic ActiVity. Cultures completely altered linkages, without compromising its high were grown in 96-well microtiter plates overnight at 37 °C transglycosylation activity. in 300 µLof2×YT medium (21), supplemented with 100 mg/L ampicillin. Cells were pelleted for 30 min at 2000g EXPERIMENTAL PROCEDURES and resuspended in 50 µL of 50 mM sodium phosphate (pH 6.5), 0.1% (w/v) SDS, 1% (w/v) Triton X-100, and 1% (w/ Oligodeoxynucleotides. Oligodeoxynucleotides were ob- v) sucrose. After incubation for4hat37°C, 50 µL of 0.01% tained from MWG Biotech (Ebersberg, Germany). The (w/v) triphenyltetrazolium chloride in 1 M NaOH was added mutagenic oligodeoxynucleotide was synthesized with 91% (25). Reducing sugars (fructose and glucose) which are of the wild type (WT) and 3% of each non-WT nucleotide released in the reaction result in formation of a red color. (NT) precursor at each degenerate position (for primer Between 5 and 15 min later, the occurrence of absorption at sequences, see the Supporting Information). 490 nm, indicating activity, was monitored with a microtiter plate reader (GENios, Tecan) and compared with that of the Bacterial Strains, Plasmids, and Culture Conditions. wild type reaction for a first selection of active candidates. Escherichia coli XL10-Gold cells (Stratagene) were used for GTFR Library Screen for Altered Transglycosylation and library construction. The recombinant plasmids were pTH275, Polymerization Products. Ninety-six-well microtiter plates harboring the full-length gtfR gene (19), and pAER100, with 200 µl of LB medium containing ampicillin (100 mg/ ′ containing a 5 -terminally truncated version (20). E. coli L) were inoculated with the GTFR mutant library stored as XL10-Gold, harboring pTH275 or its derivatives, was a cryoculture and were shaken for 2 days at 30 °C. Cells ° routinely
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