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Home , Cyclomaltodextrinase, Dextranase, Glycoside hydrolase, Invertase, Isomaltooligosaccharide, Maltase

2018; 2: 17–29

Review Article

Mary Casa-Villegas, Julia Marín-Navarro, Julio Polaina* and related with transglycosylation activity used for the production of isomaltooligosaccharides https://doi.org/10.1515/amylase-2018-0003 Abbreviations: AIG, α-glucosidase from Acremonium Received March 26, 2018; accepted May 20, 2018 implicatum; ANG, α-glucosidase from Aspergillus niger; Abstract: Isomaltooligosaccharides (IMOS) are AOG, α-glucosidase from Aspergillus oryzae; BcCITase, with health promoting properties that make them relevant CITase from Bacillus circulans T-3040; CBM, - for the pharmaceutical and food industries. IMOS have binding module; CI, cyclic isomaltooligosaccharide; ample chemical diversity achieved by different α-glucosidic CITase, cycloisomaltooligosaccharide glucanotransferase; linkages and polymerization degrees, forming linear, CMM, cyclic maltosylmaltose; CTS, cyclic ; branched and cyclic structures. Enzymatic synthesis of DP, degree of polymerization; FOS, ; these compounds can be carried out by glycoside hydrolases GH, glycoside hydrolases; GOS, ; (GHs) with transglycosylating activity. Different substrates IMOS, isomaltooligosaccharides; OPMA-N, maltogenic are used for the synthesis: combinations of amylase from Bacillus sp.; PsCITase, CITase from and , or polymeric such Paenibacillus sp. 598K; SI, ; SmuA, as or , which are converted to IMOS by a sucrose isomerase from Protaminobacter rubrum; SOG, combination of and transglucosylation. In this α-glucosidase from Schwanniomyces occidentalis; ThMA, review, the structural features of different families maltogenic amylase from Thermus sp.; XDG, α-glucosidase (GH31, GH13, GH70, GH57 and GH66) involved in IMOS from Xanthophyllomyces dendrorhous. synthesis are analysed. Focus is placed on structural traits that affect and specificity, and on the relative efficiency of transglucosylation and hydrolysis. 1 Introduction Information resulting from site-directed mutagenesis and Isomaltooligosaccharides (IMOS) are important sequence alignments complements structural data to compounds due to their health-associated properties understand the role of specific residues in the performance and potential industrial applications. IMOS could be of the . Altogether, these studies provide a frame of used as prebiotics, low calorie sweeteners or cariostatic knowledge which may be used to design new enzymes with compounds [1-4]. From a strict chemical point of view, improved properties. IMOS are linear short molecules of d- units linked by α-1,6-linkages. However, in a wider definition Keywords: Glycoside ; ; prebiotics; they are also accepted as IMOS branched and cyclic starch. glucooligosaccharides with glucose units bound by α-1,6-, α-1,3- or α-1,2-linkages even in combination with α-1,4- linkages. IMOS structure and properties depend on their *Corresponding author: Julio Polaina, Instituto de Agroquímica degree of polymerization (DP) (usually between 2 and y Tecnología de los Alimentos (IATA-CSIC), Catedrático Agustín 10 glucose units), linkage types (α-1,2, 3, 4 or 6) and the Escardino 7, Paterna (Valencia) E-46980, Spain; E-mail: jpolaina@ proportion and position of each type of linkage [5-7]. IMOS iata.csic.es properties are due to the fact that glucosidic Mary Casa-Villegas, Julia Marín-Navarro, Instituto de Agroquímica y Tecnología de los Alimentos (IATA-CSIC), Catedrático Agustín Escar- linkages, other than α-1,4, not easily hydrolysed by dino 7, Paterna (Valencia) E-46980, Spain intestinal enzymes, are suitable substrates for enzymes Julia Marín-Navarro, Department of Biochemistry and Molecular of beneficial species of the intestinal microbiota, such as Biology, Faculty of Biology, University of Valencia, Dr. Moliner 50, lactobacilli and bifidobacteria. Polymerization degree and Burjassot (Valencia) E-46100, Spain the ratio between α-1,4 and other linkages are important

Open Access. © 2018 Mary Casa-Villegas et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 4.0 License. 18 M. Casa-Villegas, et al. aspects in IMOS digestibility and by intestinal 2 Glycoside hydrolases used for microbiota [2,3,8]. The list of linear and branched IMOS described IMOS production in the literature includes , isomaltotriose, Industrial production of IMOS frequently uses starch isomaltotetraose, panose, isopanose, linear panose series, as the substrate and the combined action of different glucose-, , nigerotriose, kojibose, glycoside hydrolases (GH) enzymes with amylolytic, centose, and isomalto/maltooligosaccharides [6-22]. branching or activity, yielding a complex An important group of cyclic isomaltooligosaccharides mixture of α-glucosides [32-34]. An alternative process (CIs) are those formed by glucose units bonded only to obtaining linear or branched IMOS with defined by α-1,6-linkages, also called cyclodextrans. The DP of composition is enzymatic synthesis, using maltose or cyclodextrans ranges from 7 to 12 [4,23-27]. In addition sucrose as substrates and the catalytic action of GH to cyclodextrans, there are CIs with both α-1,6 and α-1,4- enzymes with transferase activity [6,8,9,11,12,14,16,20]. In linkages, like the cyclic maltosylmaltose (CMM) [28,29], the case of CIs, most of them are produced from dextran or with both α-1,6 and α-1,3-linkages, such as the cyclic with the concourse of cycloisomaltooligosaccharide tetrasaccharide (CTS) cyclo{-6)-α-d-Glcp-1,3-α-d-Glcp- glucanotransferases (CITases) [23,24]. 1,6-α-d-Glcp-1,3-α-d-Glcp-(1-} and its branched variants It should be pointed out that the transferases used for 4-O-α-d-glucopyranosyl-CTS and 3-O-α-isomaltosyl-CTS IMOS synthesis are not “glycosyl transferases” labelled [30,31]. A schematic representation of the different types as “GT” enzymes in the CAZy classification [35], but GH of IMOS is shown in Figure 1.

Figure 1. Chemical diversity of IMOS and related . Enzymatic synthesis of isomaltooligosaccharides 19 enzymes, in which the transferase activity is high or even (family GH31), mutant sucrose isomerase (subfamily predominant. Whereas glycosyl transferases are enzymes GH13_31) and dextransucrases (GH70). Alternatively, that require the use of nucleotide or phosphate-activated maltogenic amylase (GH13_20) and branching enzymes sugars for their function, IMOS synthesis is carried out by (GH57) have been used to produce linear IMOS from non-Leloir transferases that belong phylogenetically to , such as starch by the combined action GH enzyme families. These enzymes are characterized by of their hydrolytic and transferase activities. On the other a retaining reaction mechanism. From a structural point hand, CIs are produced by CITases (GH66) using dextran of view, in this kind of enzymes the distance between the as the substrate. In this work, structural and functional two carboxylic catalytic residues (either aspartic acid or aspects of relevant enzymes from these GH families, able glutamic acid) is around 5 Å [36]. Hydrolysis occurs in to synthesize IMOS as a result of a dual hydrolytic and two steps. In the first one (glycosylation), the acid/base transglycosylating activity, are described. residue (acting as acid) protonates the oxygen of the glycosidic linkage, while the nucleophile residue breaks the linkage by acting on the anomeric carbon and forms 3 Family GH31 a covalent enzyme-glycoside intermediate. In the second Some GH31 α- (EC 3.2.1.20) with step (deglycosylation), a water molecule, deprotonated transglycosylation activity can be used for IMOS synthesis. by the acid/base residue (acting as base), hydrolyses From a structural point of view, a representative member the covalent enzyme-glycoside intermediate and the of this family is beet α-glucosidase [44]. This glycosyl moiety is released keeping the configuration of enzyme (Fig. 2A) is composed by four protein domains: the original substrate [37,38]. Transglycosylation, as an an N-terminal β-sandwich, a (β/α) -barrel (i.e. TIM-barrel) alternative to hydrolysis, occurs when instead of water, 8 catalytic domain, a proximal C-terminal domain and a other molecule with an OH functional group, such as a distal C-terminal domain, both with a β-fold. The pocket- sugar, acts as a nucleophile on the covalent intermediate. shaped is formed mainly by loops linking the In this case, depending on the acceptor, new sugar β-strands to the α-helices of the catalytic domain and also molecules, with variable polymerization degree and/or by a long loop that bulges from the N-terminal domain new glycosidic linkages, can be synthesized. Hydrolysis called N-loop [20]. and transglycosylation reactions are performed by GH31 enzymes use maltose as the substrate to the same enzyme. The competition between the two produce IMOS with different chemical constitutions. mechanisms basically depends on the enzyme’s nature The α-glucosidases from Aspergillus niger (ANG), and the concentration of potential substrates in the Aspergillus oryzae (AOG) and Schwanniomyces reaction environment. The catalytic pocket of the enzyme occidentalis (SOG) synthesize isomaltose and panose harbours different binding subsites for each of the glycosyl as the main transglycosylation products [9,11,12,21]. An moieties composing the substrate. According to the α-glucosidase from Xanthophyllomyces dendrorhous nomenclature proposed by Davies et al. [39] the glycosidic (XDG) mainly synthesizes panose [20], while the enzyme linkage that is hydrolysed/synthesized in a catalytic cycle from Acremonium implicatum (AIG) mainly produces is located between subsites -1 and +1. Additional binding 4-α-nigerosyl glucose [14]. The transglucosylation subsites may be found depending on the substrate reaction performed by ANG occurs when an acceptor specificity of the enzyme. Subsite numbers increase molecule, either a maltose or glucose, binds to the +1/+2 positively to the reducing end of the sugar and negatively subsites, yielding panose or isomaltose, respectively to the non-reducing end. In cases where the substrate has [16,45]. The catalytic residues are Asp490 and Asp660, no reducing ends, such as sucrose, the subsite that binds corresponding to the putative acid/base and nucleophile, the glycosyl moiety that forms the enzyme-glycoside respectively. According to structural models, the +1/+2 intermediate is designated as -1. The transglycosylation subsites of ANG (Fig. 2B) would be formed by Asp225, activity of some GHs is used to synthesize other types of Thr228, Trp343, Trp453, Phe497, Arg644, Phe693, and prebiotic oligosaccharides, such as fructooligosaccharides Asn694. All these residues except Thr228, Trp343, and (FOS) [40-42] and galactooligosaccharides (GOS) [43]. Asn694 are conserved among GH31 α-glucosidases [16]. In the case of IMOS, some GHs with transglycosylation Site-directed mutagenesis studies have revealed that activity belonging to GH31, GH13, GH70, GH57 and Trp324 in SOG (equivalent to Trp343 in ANG) and Asn694 GH66 families have been use as catalytic agents (Table in ANG are involved in the transglycosylation activity of 1). Linear IMOS have been synthesized from simple these enzymes [9,16]. Mutations of Asn694 changes ANG sugars, such as maltose or sucrose, using α-glucosidases 20 M. Casa-Villegas, et al. [6] [17,58] [56] [27] [24] [23] [22] [18] [50] References [8] [14] [20] [12] [11] [9] [16] [9] - O -α-maltosyl- 2 -α-nigerosyl-maltose 2 -α-nigerosyl-maltose, 3 - O -α-nigerosyl-maltose, 2 Linear panose series; centose series; panose Linear Isomalto / maltopolysaccharides. Isomalto / Isomalto / maltopolysaccharides. Isomalto maltooligosaccharides , trehalulose and isomaltose with a with isomaltose and trehalulose Isomaltulose, R325Q: 1:0.87:1.54; R325D: 1:1.65:2.4 ratio: CIs 7-9 CIs CI-8 CI-7 - isomaltote isomaltotriose, maltotriose, Maltose, 70% type), 51% (wild percentage: IMOS traose. (W358N) 60% (W358T) and Nigerose, nigerotriose, 4-α-nigerosyl-glucose, 4-α-nigerosyl-glucose, nigerotriose, Nigerose, 3 Branched IMOS; DP 7-12 with the wild type; DP type; the wild DP 7-12 with IMOS; Branched version the mutant 10-13 with Linear IMOS DP 4 -10 IMOS Linear Main products Main maltose, 4 maltose, IMOS with DP 3-10 with IMOS Panose IMOS isomaltotriose; and isomaltose Panose, in A. niger 1:1.13:0.05 ratio panose and Isomaltose ratios: with isomaltriose and isomaltose Panose, N694A: 1:0.75:0.042; N694L: 1:0.55:0.036; N694F: 1:1.6:0.17; N694W: 1:2.5:0.36 isomaltose panose, Centose, b Sucrose and different different and Sucrose acceptors Sucrose-maltose mixture Sucrose-maltose and and Amylose maltodextrines Sucrose-glucose mixture Sucrose-glucose Dextran Starch Maltose Amylose Maltooligosaccharides Substrate 70 13_31 66 13_20 31 57 GH family a AND77013.1 AHU88292.1 R325D AAU08014.2 BAL21555.1 W358N W358T R325Q 238-247 (truncated 238-247 (truncated zone) BAA09604.1 ABY71030.1 BAD08418.1 WP_010885475.1 Protein ID Protein ALS55547.1 XP_001402053 XM_001820239 BAE20170 N694A N694L N694F N694W W324Y BAV17026.1 Dextransucrase 4,6-α-Glucanotransferase Mutant multifunctional amylase amylase multifunctional Mutant wild (SI); isomerase sucrose Mutant ID: CAF32985.1 protein type GBE mutant. Wild type protein ID: protein type Wild mutant. GBE WP_010885475.1 - gluca Cycloisomaltooligosaccharide (CITase) notransferase Multifunctional amylase (OPMA-N) amylase Multifunctional α-Glucosidase branching enzyme (GBE) enzyme branching Glycogen Enzyme Mutant α-glucosidase; wild type type wild α-glucosidase; Mutant ID: XP_001402053 protein type wild α-glucosidase; Mutant ID: BAE20170 protein 6-α-Glucosyltransferase In mutant enzymes indicates the mutated residue or the truncated zone. or the truncated residue the mutated indicates enzymes In mutant α-: sucrose, , , rafinose, , , maltose, isomaltose, nigerose, kojibiose, maltotriose, isomaltotriose. maltotriose, kojibiose, nigerose, isomaltose, maltose, lactose, galactose, rafinose, arabinose, melibiose, sucrose, α-Glucans: Weissella cibaria 10M cibaria Weissella Weissella confusa Weissella Lactobacillus reuteri 121 reuteri Lactobacillus (GTFBΔN) sp. 598K sp. Paenibacillus Protaminobacter rubrum Protaminobacter Cyclic IMOS Cyclic T3040 cepa spp., Bacillus sp. ZW2531-1 sp. Bacillus Linear IMOS Linear implicatum Acremonium Pyrococcus horikoshii Pyrococcus IMOS synthesized by different glycoside hydrolases. glycoside different by synthesized 1. IMOS Table Source a b Xanthophyllomyces dendrorhous Xanthophyllomyces niger Aspergillus oryzae Aspergillus occidentalis Schwanniomyces niger Aspergillus occidentalis Schwanniomyces 598K sp. Paenibacillus Enzymatic synthesis of isomaltooligosaccharides 21

Figure 2. (A) Crystallographic structure of a monomer of sugar beet (Beta vulgaris) α-glucosidase (PDB: 3W37) as a representative member of family GH31. N-terminal, (β/α)8-barrel, proximal and distal C-terminal domains are coloured in yellow, magenta, blue and green, respectively. Catalytic residues are depicted in light blue. (B) Homology based structural model of the catalytic centre of Aspergillus niger α-glucosidase in complex with panose. Carbohydrate-binding subsites are indicated. Catalytic residues (underlined) and other residues relevant for trans- glucosylation are highlighted. (C) Alignment of GH31 α-glucosidases sequences around relevant residues for transglucosylation (highligh- ted), marking non-conserved residues in red. α-Glucosidase source organisms: ANG, Aspergillus niger; SOG, Schwanniomyces occidentalis; AOG, Aspergillus oryzae; XDG, Xanthophyllomyces dendrorhous; AIG, Acremonium implicatum; SBG, Beta vulgaris; ROG, Ruminococcus obeum. product profile. The Asn694 is located at the β→α loop 7 and enhances the role of isomaltose as sugar acceptor of the catalytic TIM-barrel domain, and is likely involved to generate isomaltotriose [16]. A sequence alignment of in substrate binding at subsite +2. The Asn694 residue several α-glucosidases (Fig. 2C) shows that this residue has been replaced by alanine, leucine, phenylalanine or is conserved in enzymes with the same product profile tryptophan. While the wild-type AGN enzyme produced (ANG, AOG, SOG). Interestingly in XDG, which mainly panose, isomaltose and isomaltotriose with a ratio produces panose, the corresponding residue changes 1:1.13:0.05, the mutant versions changed this ratio in to alanine, in agreement with the results obtained in different ways (Table 1). In general terms, mutants the mutagenesis studies carried out with ANG. Another N694A and N694L synthesized more panose, whereas relevant position for transglycosylation is Trp343 (Fig. mutants N694F and N694W yielded more isomaltose and 2B,C). In SOG, the corresponding residue is Trp324, which isomaltotriose. Changes in Asn694 to alanine or leucine is located at the β→α loop 1 of the catalytic domain. possibly affect the orientation of Phe693, which is close to Sequence alignment of GH31 enzymes with different the +1 subsite, and may decrease the affinity for glucose substrate affinities and site-directed mutagenesis studies as glycosyl acceptor to form isomaltose. However, have shown some correlation between the presence of mutants N694W and N694F showed enhanced affinities a tyrosine at this position and a higher specificity for at subsite +2, and non-productive binding of isomaltose, the hydrolysis of α-1,4 linkages, whereas substitution probably by occupation of the +1/+2 subsites instead of by tryptophan results in increased α-1,6 specificity [46- the -1/+1. This results in decreased isomaltose hydrolysis 48]. In agreement with these observations, SOG mutant 22 M. Casa-Villegas, et al.

W324Y showed increased specificity for maltose respect 4 Family GH13 to isomaltose. On the other hand, a change in the profile of transglycosylation products was observed. GH13 enzymes share a catalytic domain with a (β/α)8- While the wild-type enzyme catalyses the formation barrel (TIM-barrel) fold, but otherwise display a wide of α-1,6 linkages between donor and acceptor sugars, a structural and functional diversity that prompted the transglycosylation product of W324Y was centose, which division of the family in more than 40 subfamilies has an α-1,2-glucosidic linkage connecting glucose and [35,51]. Enzymes belonging to subfamily 20 are involved maltose [9]. This tryptophan residue is conserved in in IMOS synthesis. This subfamily includes maltogenic ANG, AOG, SOG and XDG, all of them able to synthesize amylases (EC 3.2.1.133), neopullulanases (EC 3.2.1.135) α-1,6 linkages. In contrast, AIG with a tyrosine residue at and cyclomaltodextrinases (EC 3.2.1.54) [52]. The the equivalent position, has both α-1,3 and α-1,4-glucosyl GH13_20 maltogenic amylases characteristically have transfer activities, but not α-1,2 nor α-1,6 [14]. Altogether, transglycosylation activity. These enzymes are able to these results indicate that this residue is involved both hydrolyse α-1,4 or α-1,6-glycosidic linkages, and are also in substrate recognition and in the transglycosylation able to form transglycosylation products with α-1,3, α-1,4 product specificity. or α-1,6 linkages [53]. Compared to other GH13 α-amylases, Another GH31 member related with IMOS production which are only able to hydrolyse α-1,4-glycosidic bonds is the 6-α-glucosyltransferase from Paenibacillus sp. and have negligible transglycosylating activity, the 598K. According to its crystal structure, this is a multi- major visible structural feature of GH13_20 enzymes is modular enzyme with seven domains, four of them are the existence of an extension of about 130 residues at similar to the previously described GH31 α-glucosidases, the N-terminus of the protein sequence. This N-terminal and the remaining three are β-jelly-roll domains from stretch folds into a distinct β-sandwich domain classified carbohydrate-binding module (CBM) families – two as a CBM34 [35] and has been related to the stabilization CBM35 and one CBM61 [49]. It is an exo-acting enzyme of the dimeric structure of these enzymes (Fig. 3A). The with hydrolytic and transglycosylating activities. This N-terminal domain of one subunit covers a part of the top enzyme releases glucose from the non-reducing end of of the (β/α)8-barrel of the other subunit. The active site starch and elongates α-1,6-linked glucooligosaccharides, shaped as narrow, deep cleft, is well suited for binding yielding linear IMOS with a DP between 4-10, using cyclodextrins, which are preferred substrates of these maltooligosaccharides as substrates [50]. enzymes. Next to the active site cleft, there is another

Figure 3. (A) Crystallographic structure of the dimeric maltogenic amylase from Thermus sp. (PDB: 1SMA) as a representative member of family GH13 subfamily 20. One monomer is coloured in yellow and the second one is in green and orange. The domain highlighted in orange corresponds to the characteristic N-terminal domain of the subfamily 20 in GH13 enzymes. The and other residues involved in transglycosylation are depicted in blue. (B) Close up of the catalytic site of the maltogenic amylase 1SMA. The catalytic triad (underlined) composed by D328 (nucleophile), E357 (acid/base) and D424 (transition state stabilizer) and residues with a role in transglucosylation are indicated. W359 and E332 are homologous to W358 and E331, respectively, in OPMA-N from Bacillus sp. Enzymatic synthesis of isomaltooligosaccharides 23 space, absent in other subfamilies, suitable for binding a residues that is a key player in the catalytic mechanism . This space likely hosts an acceptor molecule controlling/modulating the entrance/exit of the substrate/ in the transglycosylation reaction and also allows the product. SIs have an isomerization motif (RLDRD) [55], binding of a branched for the hydrolysis also called a - [56]. A single mutation of an α-1,4 or α-1,6- [53,54]. in SmuA (R325N or R325D) at the fructose-binding site In the maltogenic amylase OPMA-N from Bacillus changed the enzyme product profile. While the wild-type sp., also called multifunctional amylase-N, which is able enzyme, using a sucrose-glucose mixture as substrate, to use starch as substrate to generate isomaltotriose and synthesized preferentially isomaltulose, mutants isomaltotetraose, the highly conserved Trp358 residue R325N and R325D yielded isomaltose preferentially has been specifically related to transglycosylation [56]. Located at the +1 subsite, Arg325 may contribute to activity. Trp358 is near to the catalytic triad, composed further stabilization of the fructose ring for isomaltulose by nucleophile Asp327, acid/base catalytic Glu356, and synthesis [55]. The replacement of positively charged the stabilizer of the catalytic intermediate, Asp423, in a arginine residue with either or aspartic acid loop of the TIM-barrel, at subsite +2. Figure 3B shows the increased the average distance between the mutated active centre of Thermus sp. maltogenic amylase (ThMA), amino acid residue and the fructosyl moiety, decreasing homologous to OPMA-N. Several mutations of Trp358 fructose affinity at the active site. In the proposed reaction show the involvement of this residue in transglycosylation mechanism for mutant enzymes, sucrose is cleaved into activity. Using the wild-type OPMA-N with starch as glucose and fructose, the fructose moiety is released, and the substrate, around 50% of the products are IMOS finally a second molecule of glucose acting as acceptor is (isomaltotriose and isomaltotetrose). Mutations with bound to the glucosyl moiety through an α-1,6-glycosidic negatively charged residues (W358D, W358E) or polar linkage [56]. residues (W358N, W358T), enhanced IMOS formation. However, positively charged residues (W358K, W358R) abolished transglycosylation, while keeping hydrolytic 5 Family GH70 activity [22]. Polar residues at subsite +2 likely provide The family GH70 includes several type of enzymes a better orientation to acceptor sugars, whereas the with transferase activity: 4,6-α-glucanotransferases, introduction of negative or positive charges at this position 4,3-α-glucanotransferases, dextransucrases, probably move catalytic Glu356 away from glucosyl alternansucrases, mutansucrases, reuteransucrases and acceptors, facilitating or impeding transglycosylation in α-1,2-branched dextransucrases [35]. These enzymes have one case or the other. A conserved glutamic acid residue been related to IMOS synthesis. in maltogenic amylases (Glu331 in OPMA-N) placed GH70 4,6-α and 4,3-α-glucanotransferases are between the N-domain of a monomer and the (β/α) -barrel 8 able to hydrolyse α-1,4-glycosidic linkages from domain of the adjacent monomer, has also been related to maltooligosacaccharides and starch and perform transglycosylating activity. This glutamate residue could transglycosylation yielding α-1,6 or α-1,3-glycosidic play an important role in the binding of oligosaccharide linkages, respectively. Most 4,6-α-glucanotransferases acceptors [54]. In the maltogenic amylase from Thermus produce linear isomalto-/maltopolysaccharides and sp. (ThMA), the corresponding Glu332 residue (located isomalto-/maltooligosaccharides [17,57,58]. In contrast, at hydrogen bonding distance to a modelled maltose GH70 glucansucrases use sucrose but not starch as the molecule) was mutated to histidine. The result was a substrate. These enzymes also catalyse transglycosylation drastic reduction of α-1,6 transglycosylation products of the glucosyl moiety from sucrose to another acceptor without affecting the hydrolytic activity [53]. through α-1,2, α-1,3, α-1,4 or α-1,6 linkages. Within this Mutant versions of a sucrose isomerase (SI) from group, dextransucrases and mutansucrases specifically Protaminobacter rubrum (SmuA) belonging to GH13_31 yield α-1,6 or α-1,3-glycosydic linkages, respectively [6,8]. subfamily has been used for isomaltose synthesis. Wild- GH70 enzymes show wide differences in the length type GH13_31 SI catalyses the isomerization of sucrose to of their primary structure, reflecting variations around a isomaltulose or trehalulose. GH13_31 SIs have the typical common fold formed by five-domains designated A, B, C, topology of GH13 enzyme structures with a three domain IV and V (Fig. 4A). Domains A, B and C are also found in organization, the (β/α) -barrel catalytic domain at the 8 related GH13 enzymes, which together with families GH70 N-terminus that includes a loop rich subdomain and the and GH77 constitute the clan GH-H. Domains IV and V are antiparallel β-sandwich domain at C-terminus. GH13_31 unique to the GH70 family [59]. At the interface of the A and SI has an aromatic clamp formed by two phenylalanine 24 M. Casa-Villegas, et al.

Figure 4. (A) Crystallographic structure of the common fold of the GH70 family represented by the from Lactobacillus reuteri 180 GTF180-ΔN (PDB: 3HZ3). The five domains of the enzyme: A, B, C, IV and V are coloured in blue, green, magenta, yellow and red, respectively. The catalytic triad is depicted in orange. (B) Overlaying of the catalytic site of a 4,6-α-glucanotransferase from Lactobacillus reuteri 121 (PDB: 5JBD), in blue; a mutansucrase from Streptococcus mutans (PDB: 3AIB), in green; and a dextransucrase from Leuconostoc citreum NRRL B-1299 (PDB: 5NGY) in yellow. Residues of the catalytic triad are underlined. Sucrose, located at the catalytic site, is depicted in orange. Carbohydrate-binding subsites are indicated.

B domains, several residues that are strictly conserved in open architecture of the active site of glucansucrases may GH13 and GH70 enzymes surround a pocket that harbours explain their ability to form branched α-glucans whereas subsites -1 and +1 of the active site [17]. There is evidence that most 4,6-α-glucantrasferases, only synthesize linear ones GH70 enzymes evolved from GH13 α-amylases with GH70 [17]. 4,6-α-glucanotransferases representing the intermediate Two residues at subsite +1 have been involved in the link to other GH70 enzymes [17]. Rearrangement in three transglycosylating activity and product specificity of loops (A1, B, A2) near the active site explain the evolution glucanotransferases (Fig. 4B). The importance of these from GH13 to GH70 enzymes and associated functional residues has been shown by site-directed mutagenesis consequences. In GH70 4,6-α-glucanotransferases, loops of Lactobacillus reuteri 4,6-α-glucanotransferase. The A1 and B have no equivalent in GH13 α-amylases that only wild-type enzyme converts to linear hydrolyse α-1,4 linkages of starch. These loops fold over isomalto-/maltooligosaccharides with a relatively high the donor side of the binding groove, forming a tunnel- percentage of α-1,6 linkages (82% of the total product). like structure that allows 4,6-α-glucanotransferases In contrast, mutant versions of the enzyme (Y1055G, to sizing the glycosyl intermediate for subsequent K1128A, K1128N, K1128H and K1128W) mainly display transglycosylation, in a processive mode of action. endo-α-1,4-glycosidase activity with drastically decreased Thus, the structure evolution converted starch- transglycosylating activity. Only around 1-8% of the total hydrolysing enzymes into starch-modifying enzymes with product mixture linkages were α-1,6 [17]. On the other hand, transglycosylating activity. In glucansucrases, compared residue divergences at these positions, where tyrosine with 4,6-α-glucantrasferases, loops A1 and B are shorter and lysine are replaced by tryptophan and glutamine, and create a more open arrangement. In contrast, the loop respectively, in mutansucrases and dextransucrases (Fig. A2 is longer, blocking access to donor subsites beyond -1. 4B), has been related to different product specificity. In As a result, glucansucrases use specifically sucrose, but GH70 glucansucrases, structural studies suggest that a not starch, as sugar donor for polymerization. The more residue at the +1 subsite determines the specificity of the Enzymatic synthesis of isomaltooligosaccharides 25 linkage type that is formed upon transglucosylation by (subsite -1) attacks the C1 atom of the glucose unit, creating directing the relative orientation of glucosyl donor and a covalent intermediate between the enzyme and substrate. acceptor. In Streptococcus mutans the aspartic acid that In the branching process, the cleaved product remains in the occupies this position at mutansucrases, is replaced by a groove mainly due to interaction with Tyr236 in the flexible threonine in the dextransucrase [60]. loop that protrudes into the +1 subsite. At the last step of the branching process, the OH group attached to the C6 glucose at the +1 subsite carries out the nucleophilic attack 6 Family GH57 on the enzyme-glycosyl covalent intermediate, resulting in formation of the branched with an α-1,6-glycosidic bond. In Enzymes from the GH57 family with branching activity (EC this process the Trp22 residue, which is at the bottom region 2.4.1.18) catalyse the formation of α-1,6 branching points, by of the groove, far from the catalytic site, plays an important cleaving an α-1,4-glycosidic bond in amylose and transferring role presumably by binding the non-reducing end of the the excised oligosaccharide to a chain, via an α-1,6 substrate in the groove. When the Trp22 from Pyrococcus bond. The overall structure of these enzymes, composed by horikoshii branching enzyme was changed to an alanine, three protein domains, shows a triangular shape. Domain A the mutant enzyme completely lost the activity [18]. is a (β/α) -barrel. An insertion of two or three α-helices that 7 Tyr236 in the flexible loop is conserved among bulges out from domain A constitutes domain B. Domain other GH57 enzymes [18,61,62]. In Thermus thermophilus C, in C-terminal position, is made by a helical bundle branching enzyme, a mutation of this tyrosine residue (Fig. 5A). The active-site cleft is a long groove located at (Y236A) abolished all branching activity, with a the interface of domains A and C. The groove is decorated concomitant 10-fold increase in hydrolysis activity [61]. In with aromatic residues on the surface that possibly play a mutant enzyme from Pyrococcus horikoshii with a shorter a role in substrate recognition and binding, and lead the loop, made by deleting amino acid residues 238-247 but substrate to the reactive residues. GH57 enzymes have a keeping Tyr236, the branching activity was decreased. flexible loop at the top region of the active site groove that Furthermore, while in the wild-type enzyme the DP of the is essential for the branching activity [18,61,62]. Because of reaction products ranged from 7 to 12, in the loop-deleted the mobility freedom of this region, it is not visible in many mutant the size of the products were slightly longer than crystallographic structures, but it has been solved for the those of the wild-type enzyme, increasing from 10 to 13 Thermococcus kodakaraensis branching enzyme (Fig. 5B). units [18]. In the catalytic mechanism of a member of the family from Pyrococcus horikoshii [18], the nucleophilic residue Glu185

Figure 5. (A) Crystallographic structure of a branching enzyme from Thermococcus kodakaraensis KOD1 (PDB: 3N8T) as a representative member of GH57 family. Domains A (corresponding to the (β/α)7-barrel), B and C are indicated in orange, blue and green, respectively. Catalytic residues are coloured in light blue. (B) Superposition of the structures of the branching enzyme from T. kodakaraensis (PDB: 3N98) and that from Pyrococcus horikoshii (PDB: 5WU7) in orange and green, respectively. The flexible loop covering the active site groove, only solved in the 3N98 structure, is coloured in blue. Catalytic residues (E183, nucleophile and D354, acid/base) are underlined and highligh- ted together with residues involved in transglucosylation. Molecules of glycerol (gray) and glucose (purple) in complex with 3N98 structure appear located at the catalytic groove. 26 M. Casa-Villegas, et al.

7 Family GH66 +1 functions as an acceptor of the transglucosylation reaction, completing the cyclization and releasing a CI Family GH66 contains CITases (EC2.4.1.248) and dextranases of n units [24]. The DP of the CIs synthesized by different (EC3.2.1.11). Whereas GH66 dextranases produce only IMOS CITases is attributed to the second sugar-binding site, and by dextran hydrolysis, CITases produce predominantly specifically to the residue located at subsite -5 [23,24]. CIs but also small amounts of IMOS. CITases catalyse the Thus, the CITase from Paenibacillus sp. 598K (PsCITase), intramolecular transglucosylation of dextran to produce which mainly synthesizes CI-7 has Gln502 at this subsite, CIs with a DP equal or higher than seven. while the CITase from Bacillus circulans T-3040 (BcCITase), CITases structure consists of four domains (Fig. 6A): a which mainly synthesizes CI-8, has a phenylalanine catalytic (β/α)8-barrel domain and three β-domains. One of residue at the equivalent position (Phe501). The these is a family CBM35 (CBM35-1), with a β-jelly-roll fold, conformation of Gln502 in PsCITase allows the sugar chain which protrudes from the catalytic domain and possesses to interact with Trp515 at subsite -6. However, the bulkier two sugar-binding sites. The first is the canonical sugar- Phe501 side chain forces the sugar chain to circumvent binding site located on the top of the domain, where most this position, reaching Trp514 at subsite -8 (Fig. 6B) [23]. CBM35s recognize their cognate sugars. The second site is In CIs synthesis, the first sugar binding site at CBM35-1 located at the edge of one β-sheet, recognizing the non- plays an important role recognizing the substrate. In reducing end glucose of α-1,6-linked glucans [23,24]. BcCITase, the first sugar binding site mainly recognizes Once the α-glucan is bound to the enzyme’s one glucose moiety through stacking interactions with catalytic site, the α-glucosidic linkage between the two Trp506 and hydrogen bonds with His439, Asn539 and at positions -1 and +1 is cleaved and the glucose Gln450. His439, Trp506, and Asn539 are highly conserved at subsite -1 is held covalently bound to the catalytic among CBM35s [24]. In PsCITase, Trp507 and Trp515 nucleophile as an enzyme-substrate intermediate. residues located at CBM35-1 at first and second sugar Subsequently, the glucose residue at the non-reducing end binding sites, respectively, and equivalents to Trp506 and of the cleaved substrate, located at a distance of n residues, Trp514 in BcCITase, were mutated to alanine. The mutants is released from its place at position -n and transferred to (W507A and W515A) mainly produced CIs of seven units as subsite +1 by folding into a circular conformation. In this in the wild-type enzyme but their overall production was operation, the glucose residue that is relocated at subsite reduced approximately by 25% and 65%, respectively [23].

Figure 6. (A) Crystallographic structure of a CITase from Paenibacillus sp. 598K (PDB: 5X7G) as a representative member of GH66 family. The catalytic (β/α)8 domain and the three β-domains are in green, blue, orange and yellow, respectively. The yellow β-domain corresponds to CBM35-1 domain. Highlighted residues conform sugar binding sites 1 (purple) and 2 (blue). Catalytic residues are depicted in red. (B) Super- position and close up view of the catalytic centre, of the CITase from Paenibacillus sp. 598K (PDB: 5X7H) (green) and CITAse from Bacillus cir- culans T3040 (PDB: 3WNN) (orange). Sugar binding sites and catalytic residues are indicated. The structures 5X7H and 3WNN are in complex with isomaltoheptaose (light blue) and isomaltooctaose (brown), respectively. Carbohydrate-binding subsites are indicated. Enzymatic synthesis of isomaltooligosaccharides 27

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