Insights into Broad-Specificity Starch Modification from the Crystal Structure of Limosilactobacillus Reuteri NCC 2613 4,6-α-Glucanotransferase GtfB

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly “open” and thus suitable as broad-specificity starch-converting enzymes

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-d-glucosylated at the steviol C-19 β-d-glucosyl moiety (55% conversion). The main product is a mono-(α1 → 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 → 3)- and (α1 → 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties

Goat Milk Oligosaccharides:Their Diversity, Quantity, and Functional Properties in Comparison to Human Milk Oligosaccharides

Human milk is considered the golden standard in infant nutrition. Free oligosaccharides in human milk provide important health benefits. These oligosaccharides function as prebiotics, immune modulators, and pathogen inhibitors and were found to improve barrier function in the gut. Infant formulas nowadays often contain prebiotics but lack the specific functions of human milk oligosaccharides (hMOS). Milk from domesticated animals also contains milk oligosaccharides but at much lower levels and with less diversity. Goat milk contains significantly more oligosaccharides (gMOS) than bovine (bMOS) or sheep (sMOS) milk and also has a larger diversity of structures. This review summarizes structural studies, revealing a diversity of up to 77 annotated gMOS structures with almost 40 structures fully characterized. Quantitative studies of goat milk oligosaccharides range from 60 to 350 mg/L in mature milk and from 200 to 650 mg/L in colostrum. These levels are clearly lower than in human milk (5-20 g/L) but higher than in other domesticated dairy animals, e.g., bovine (30-60 mg/L) and sheep (20-40 mg/L). Finally, the review focuses on demonstrated and potential functionalities of gMOS. Some studies have shown anti-inflammatory effects of mixtures enriched in gMOS. Goat MOS also display prebiotic potential, particularly in stimulating growth of bifidobacteria preferentially. Although functional studies of gMOS are still limited, several structures are also found in human milk and have known functions as immune modulators and pathogen inhibitors. In conclusion, goat milk constitutes a promising alternative source for milk oligosaccharides, which can be used in infant formula

Potential Dental Biofilm Inhibitors: Dynamic Combinatorial Chemistry Affords Sugar-Based Molecules that Target Bacterial Glucosyltransferase

We applied dynamic combinatorial chemistry (DCC) to find novel ligands of the bacterial virulence factor glucosyltransferase (GTF) 180. GTFs are the major producers of extracellular polysaccharides, which are important factors in the initiation and development of cariogenic dental biofilms. Following a structure-based strategy, we designed a series of 36 glucose- and maltose-based acylhydrazones as substrate mimics. Synthesis of the required mono- and disaccharide-based aldehydes set the stage for DCC experiments. Analysis of the dynamic combinatorial libraries (DCLs) by UPLC-MS revealed major amplification of four compounds in the presence of GTF180. Moreover, we found that derivatives of the glucose-acceptor maltose at the C1-hydroxy group act as glucose-donors and are cleaved by GTF180. The synthesized hits display medium to low binding affinity (KD values of 0.4–10.0 mm) according to surface plasmon resonance. In addition, they were investigated for inhibitory activity in GTF-activity assays. The early-stage DCC study reveals that careful design of DCLs opens up easy access to a broad class of novel compounds that can be developed further as potential inhibitors

Enzymatic glucosylation of polyphenols using glucansucrases and branching sucrases of glycoside hydrolase family 70

Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.</p

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri

Previously, we have shown that the glucansucrase GtfA-ΔN 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-ΔN 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-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-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-ΔN used α-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-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-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

Trans-α-glucosylation of stevioside by the mutant glucansucrase enzyme Gtf180-ΔN-Q1140E improves its taste profile

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient alpha-glucosylation of stevioside using the mutant glucansucrase Gtf180-Delta N-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the alpha-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties

Actinomycete integrative and conjugative elements

This paper reviews current knowledge on actinomycete integrative and conjugative elements (AICEs). The best characterised AICEs, pSAM2 of Streptomyces ambofaciens (10.9 kb), SLP1 (17.3 kb) of Streptomyces coelicolor and pMEA300 of Amycolatopsis methanolica (13.3 kb), are present as integrative elements in specific tRNA genes, and are capable of conjugative transfer. These AICEs have a highly conserved structural organisation, with functional modules for excision/integration, replication, conjugative transfer, and regulation. Recently, it has been shown that pMEA300 and the related elements pMEA100 of Amycolatopsis mediterranei and pSE211 of Saccharopolyspora erythraea form a novel group of AICEs, the pMEA-elements, based on the unique characteristics of their replication initiator protein RepAM. Evaluation of a large collection of Amycolatopsis isolates has allowed identification of multiple pMEA-like elements. Our data show that, as AICEs, they mainly coevolved with their natural host in an integrated form, rather than being dispersed via horizontal gene transfer. The pMEA-like elements could be separated into two distinct populations from different geographical origins. One group was most closely related to pMEA300 and was found in isolates from Australia and Asia and pMEA100-related sequences were present in European isolates. Genome sequence data have enormously contributed to the recent insight that AICEs are present in many actinomycete genera. The sequence data also provide more insight into their evolutionary relationships, revealing their modular composition and their likely combined descent from bacterial plasmids and bacteriophages. Evidence is accumulating that AICEs act as modulators of host genome diversity and are also involved in the acquisition of secondary metabolite clusters and foreign DNA via horizontal gene transfer. Although still speculative, these AICEs may play a role in the spread of antibiotic resistance factors into pathogenic bacteria. The novel insights on AICE characteristics presented in this review may be used for the effective construction of new vectors that allows us to engineer and optimise strains for the production of commercially and medically interesting secondary metabolites, and bioactive proteins

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-D-glucosylated at the steviol C-19 β-D-glucosyl moiety (55% conversion). The main product is a mono-(α1 → 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 → 3)- and (α1 → 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties