Structure of the ?-1,6/?-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121.
ABSTRACT: The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched ?-glucans with both ?-1,6- and ?-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-?N, a 118?kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined at 3.6?Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-?N has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-?N active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an ?-1,6/?-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128-1136 in GTFA-?N) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.
Project description:Previously, we have shown that the glucansucrase GtfA-?N enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-?N with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-?N used both ?-D-Glcp-catechol and ?-D-Glcp-(1?4)-?-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-?N used ?-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-?N from L. reuteri 180 and GtfML1-?N from L. reuteri ML1, used ?-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.
Project description:The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes.
Project description:Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-D-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-D-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.
Project description:The glucansucrase GTFA of Lactobacillus reuteri 121 produces an ?-glucan (reuteran) with a large amount of alternating (?1???4) and (?1???6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an ?-glucan with (?1???4) and (?1???6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 (970DGKGYKGA977) and helix ?4 (1083VSLKGA1088) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (?1???4) and (?1???6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3…) together form a distinct physicochemical micro-environment that determines the alternating (?1???4) and (?1???6) linkages synthesis in GTFA.
Project description:Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of ?-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing ?-glucan synthesis by mutational engineering of the Gtf180-?N enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-?N mutants (L938F, L981A, and N1029M) with an impaired ?-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-?-D-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-?N protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired ?-glucan synthesis, thus yielding mutants with an improved glycosylation potential.
Project description:Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of ?-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu(940) in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-?N in complex with maltose. Herein, we show that mutations in Leu(940) of wild-type GTF180-?N all caused an increased percentage of (?1?6) linkages and a decreased percentage of (?1?3) linkages in the products. ?-Glucans with potential different physicochemical properties (containing 67-100% of (?1?6) linkages) were produced by GTF180 and its Leu(940) mutants. Mutant L940W was unable to form (?1?3) linkages and synthesized a smaller and linear glucan polysaccharide with only (?1?6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (?1?3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu(940) in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.
Project description:Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into ?-glucan polysaccharides with (?1???2)-, (?1???3)-, (?1???4)- and/or (?1???6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-?-glucanotransferase activity, cleaving (?1???4)-linkages and synthesizing new (?1???6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-?-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-?-glucanotransferase, converting amylose into a new type of ?-glucan with alternating (?1???3)/(? 1???4)-linkages and with (?1???3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of ?-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes.
Project description:?N(123)-glucan-binding domain-catalytic domain 2 (?N(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays ?-(1?2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of ?-(1?2) linkages. The crystal structure of the apo ?-(1?2) branching sucrase ?N(123)-GBD-CD2 was solved at 1.90 ? resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-?N and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-?N and ?N(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ?N(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-?N showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ?N(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of ?-(1?2) branches.
Project description:Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.
Project description:Lactic acid bacteria (LAB) are known to produce large amounts of ?-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these ?-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of ?-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-?-glucanotransferases (4,6-?-GTs) that represent novel evolutionary intermediates between the family GH13 and "classical GH70 enzymes". These enzymes are not active on sucrose; instead, they use (?1 ? 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel ?-glucans by introducing linear chains of (?1 ? 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure-function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes.