University of Groningen Glycosidic Bond Specificity of Glucansucrases

University of Groningen Glycosidic bond specificity of glucansucrases Leemhuis, Hans; Pijning, Tjaard; Dobruchowska, Justyna M.; Dijkstra, Bauke W.; Dijkhuizen, Lubbert Published in: Biocatalysis and Biotransformation DOI: 10.3109/10242422.2012.676301 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: 2012 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leemhuis, H., Pijning, T., Dobruchowska, J. M., Dijkstra, B. W., & Dijkhuizen, L. (2012). Glycosidic bond specificity of glucansucrases: on the role of acceptor substrate binding residues. Biocatalysis and Biotransformation, 30(3), 366-376. DOI: 10.3109/10242422.2012.676301 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). 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: 11-02-2018 Biocatalysis and Biotransformation, May–June 2012; 30(3): 366–376 ORIGINAL ARTICLE Glycosidic bond specifi city of glucansucrases: on the role of acceptor substrate binding residues HANS LEEMHUIS 1 , TJAARD PIJNING 2 , JUSTYNA M. DOBRUCHOWSKA 1 , BAUKE W. DIJKSTRA 2 & LUBBERT DIJKHUIZEN 1 1 Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, The Netherlands and 2 Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, The Netherlands Abstract Many lactic acid bacteria produce extracellular a -glucan polysaccharides using a glucansucrase and sucrose as glucose donor. The structure and the physicochemical properties of the a -glucans produced are determined by the nature of the glucansucrase. Typically, the a -glucans contain two types of a -glycosidic linkages, for example, (a 1 - 2), (a 1 - 3), (a 1- 4) or (a 1 - 6), which may be randomly or regularly distributed. Usually, the a -glucan chains are also branched, which gives rise to an additional level of complexity. Even though the fi rst crystal structure was reported in 2010, our current understanding of the structure– function relationships of glucansucrases is not advanced enough to predict the a -glucan specifi city from the sequence alone. Nevertheless, based on sequence alignments and site-directed mutagenesis, a few amino acid residues have been identifi ed as being important for the glycosidic bond specifi city of glucansucrases. A new development in GH70 research was the identifi cation of a cluster of a -glucan disproportionating enzymes. Here, we discuss the current insights into the structure– function relationships of GH70 enzymes in the light of the recently determined crystal structure of glucansucrases. Keywords: alternansucrase , dextransucrase , glycoside hydrolase , reuteransucrase , lactic acid bacteria , protein engineering For personal use only. Introduction Glucansucrases Many bacteria form a biofi lm matrix to protect them GSs are a -glucan synthesizing enzymes of the glyco- from environmental stress and to keep extracellular side hydrolase family 70 (GH70) (Cantarel et al. digestive enzymes in the vicinity of the cells, thus 2009), a group of extracellular enzymes produced ensuring that the extracellular enzymes are not pro- predominantly by Lactobacillales bacteria (van Hijum viding nutrients to competing organisms (Flemming et al. 2006). These enzymes are also known as glu- & Wingender 2010; Walter et al. 2008; Kaditzky cosyltransferases, abbreviated as ‘ GTF ’ , because et al. 2008). Moreover, biofi lms are key to surface they synthesize a -glucan polymers using the glucose colonization, such as tooth enamel colonization by unit of sucrose. GH70 enzymes are evolutionarily Streptococcus species (Nobbs et al. 2009; Freitas et al. closely related to the GH13 and GH77 enzymes and 2011). Biofi lms are typically composed of polymeric together they form the GH-H clan according to the Biocatal Biotransformation Downloaded from informahealthcare.com by University of Groningen on 06/27/12 compounds, for example, carbohydrates, proteins Carbohydrate-Active Enzymes (CAZy) (http://www. and DNA. Several lactic acid bacteria form biofi lms cazy.org) (Cantarel et al. 2009) classifi cation system. that are largely composed of a -glucan polymers, The GH-H clan encompasses a large group of which are synthesized from sucrose by an extracel- enzymes acting on a -glucan carbohydrates such as lular glucansucrase (GS) (Schwab et al. 2007; Anwar starch, glycogen, pullulan, sucrose, etc. Well known et al. 2010; Leathers & Bischoff 2011). In this review, enzymes of the GH-H clan are a -amylase, branching we focus on the structure – function relationships of enzyme, cyclodextrin glucanotransferase, pullula- GSs and the structural properties of the a -glucan nase and amylomaltase (MacGregor et al. 2001; produced by this class of enzymes. Kuriki & Imanaka 1999; van der Maarel et al. 2002; Correspondence: Lubbert Dijkhuizen, Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, The Netherlands. Tel: 31 - 50-3632150. Fax: 31 - 50-3632154. E-mail: [email protected] ISSN 1024-2422 print/ISSN 1029-2446 online © 2012 Informa UK, Ltd. DOI: 10.3109/10242422.2012.676301 Glycosidic bond specifi city of glucansucrases 367 Kelly et al. 2009b; Palomo et al. 2011). GSs are specifi cities have been observed among GSs, though intensively studied as they are found in many probi- typically GSs form two types of glycosidic linkages otic bacteria, and they are thought to contribute to only, for example, (a 1 - 3) and (a 1 - 6), (a 1 - 4) and the probiotic properties via the a -glucan products (a 1 - 6) or (a 1 - 2) and (a 1 - 6). The next level of com- they make. Moreover, there are indications that the plexity arises from the fact that the a -glucan prod- a -glucan products of GSs have prebiotic activities ucts formed are branched, which implies that the and may act as anti-adhesives hampering coloniza- acceptor subsites not only can accommodate the tion by harmful bacteria (Wang et al. 2010). non-reducing end of a glucan chain, resulting in chain elongation, but also the glucan chain itself to create a branch. How GSs can specifi cally recognize The catalytic cycle of glucansucrases and orient acceptor molecules to create highly com- plex a -glucans is a very intriguing question. Although Catalysis by GSs is generally believed to be a modelling of acceptor molecules in the active site has two-step process, which starts with the binding provided some clues (Vujicic-Zagar et al. 2010), our of the substrate sucrose and the cleavage of the current understanding of the glycosidic bond speci- (a 1 - 2)-glycosidic linkage between the glucose and fi city of GSs is only poorly developed. fructose moieties of sucrose yielding a covalent GSs can also transfer the glycosyl intermediate glycosyl-enzyme intermediate. In the catalytic to a wide variety of hydroxyl-group containing mol- machinery, Asp1025 acts as the nucleophile forming ecules in a so called acceptor reaction, which is use- a covalent linkage with the glycosyl moiety, and ful in (chemo-) enzymatic synthesis, as it prevents Glu1063 acts as a general acid/base catalyst (amino all kind of diffi culties regarding stereo- and regio- acid numbering according to GTF180 of Lactobacillus specifi city associated with the chemical synthesis of reuteri 180, unless stated otherwise). Following the (hybrid) carbohydrate compounds (Hanson et al. departure of fructose from the ϩ 1 acceptor subsite, 2004; Homann et al. 2009a,b; Leemhuis et al. an acceptor substrate can enter the acceptor subsites 2010). The acceptor substrate promiscuity of GSs to attack the covalent glycosyl-enzyme intermediate. has been used to elongate short oligosaccharides The resulting product again has an a -glycosidic link- (Cote & Sheng 2006), to label dextran with 13 C- age. The existence of the covalent glycosyl-enzyme glucose (Mukasa et al. 2001), to glucosylate gentio- intermediate has not yet been demonstrated experi- biose (Cote 2009a), raffi nose (Cote 2009b), mentally for a GH70 enzyme; however, its existence nigero-oligosaccharides (Komatsu et al. 2011), fl a- has convincingly been demonstrated by protein vonoids (Bertrand et al. 2006), hydroquinones (Seo crystallography for the GH13 enzymes, cyclodextrin et al. 2009), salicin and phenol (Seo et al. 2005), For personal use only. glucanotransferase (Uitdehaag et al. 1999) and primary alcohols (Seibel et al. 2006a), methyl, alkyl, amylosucrase (Jensen et al. 2004) and for the GH77 butyl and octyl-D-glucopyranosides (de Segura enzyme, amylomaltase (Barends et al. 2007), et al. 2006; Richard et al. 2003; Kim et al. 2009) which also belong to the GH-H clan of glycoside and to synthesize branched thiooligosaccharides hydrolases. (Hellmuth et al. 2007). Another well-known product of the acceptor reaction is the sucrose isomer leucrose

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