Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g. in food, health and as biomaterials. Originally, the GH70 family was established only for glucansucrase enzymes of lactic acid bacteria that catalyze the synthesis of α-glucan polymers from sucrose. In recent years, we have identified 3 novel subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD), inactive on sucrose but converting starch/maltodextrin substrates into novel α-glucans. These novel starch-acting enzymes considerably enlarge the panel of α-glucans that can be produced. They also represent very interesting evolutionary intermediates between sucrose-acting GH70 glucansucrases and starch-acting GH13 α-amylases. Here we provide an overview of the repertoire of GH70 enzymes currently available with focus on these novel starch-acting GH70 enzymes and their biotechnological potential. Moreover, we discuss key developments in the understanding of structure-function relationships of GH70 enzymes in the light of available three-dimensional structure structures, and the protein engineering strategies that were recently applied to expand their natural product specificities.</p

The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D Lombard et al. (2014) H/ Machius et al. (1995) C NMR spectroscopy and enzymatic degradation studies. RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient

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

Biochemical characterization of two GH70 family 4,6-α-glucanotransferases with distinct product specificity from Lactobacillus aviarius subsp. aviarius DSM 20655

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the "fingerprint" Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655, a second GtfB-like protein (GtfY), containing the canonical GtfB Tyr residue in motif III, was located directly upstream of GtfX. Biochemical characterization revealed that both GtfX and GtfY showed GtfB-like 4,6-α-GT activity, cleaving (α1→4) linkages and catalyzing the synthesis of (α1→6) linkages. Nonetheless, they differ in product specificity; GtfY only synthesizes consecutive (α1→6) linkages, yielding linear α-glucan products, but GtfX catalyzes the synthesis of (α1→6) linkages predominantly at branch points (22%) rather than in linear segments (10%). The highly branched α-glucan produced by GtfX from amylose V is resistant to digestion by α-amylase, offering great potential as dietary fibers

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

The glycemic carbohydrates we consume are currently viewed in an unfavorable light in both the consumer and medical research worlds. In significant part, these carbohydrates, mainly starch and sucrose, are looked upon negatively due to their rapid and abrupt glucose delivery to the body which causes a high glycemic response. However, dietary carbohydrates which are digested and release glucose in a slow manner are recognized as providing health benefits. Slow digestion of glycemic carbohydrates can be caused by several factors, including food matrix effect which impedes α-amylase access to substrate, or partial inhibition by plant secondary metabolites such as phenolic compounds. Differences in digestion rate of these carbohydrates may also be due to their specific structures (e.g. variations in degree of branching and/or glycosidic linkages present). In recent years, much has been learned about the synthesis and digestion kinetics of novel α-glucans (i.e. small oligosaccharides or larger polysaccharides based on glucose units linked in different positions by α-bonds). It is the synthesis and digestion of such structures that is the subject of this review

Crystal Structure of 4,6-α-Glucanotransferase Supports Diet-Driven Evolution of GH70 Enzymes from α-Amylases in Oral Bacteria

Food processing and refining has dramatically changed the human diet, but little is known about whether this affected the evolution of enzymes in human microbiota. We present evidence that glycoside hydrolase family 70 (GH70) glucansucrases from lactobacilli, synthesizing α-glucan-type extracellular polysaccharides from sucrose, likely evolved from GH13 starch-acting α-amylases, via GH70 4,6-α-glucanotransferases. The crystal structure of a 4,6-α-glucanotransferase explains the mode of action and unique product specificity of these enzymes. While the α-amylase substrate-binding scaffold is retained, active-site loops adapted to favor transglycosylation over hydrolysis; the structure also gives clues as to how 4,6-α-glucanotransferases may have evolved further toward sucrose utilization instead of starch. Further supported by genomic, phylogenetic, and in vivo studies, we propose that dietary changes involving starch (and starch derivatives) and sucrose intake were critical factors during the evolution of 4,6-α-GTs and glucansucrases from α-amylases, allowing oral bacteria to produce extracellular polymers that contribute to biofilm formation from different substrates

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures

Draft Genome Sequence of Lactobacillus reuteri 121, a Source of α-Glucan and β-Fructan Exopolysaccharides

The probiotic bacterium Lactobacillus reuteri 121 is a well-known producer of diverse homoexopolysaccharides (α-glucans and β-fructans) from sucrose and maltodextrins/starches of interest for food applications. Here, we report the draft genome sequence of this strain, with a focus on carbohydrate-active enzymes

Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database:The Lactobacillus reuteri NCC 2613 GtfB

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesizing new (α1 → 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 → 4) sequences interspersed with (α1 → 6) linkages, and (α1 → 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 → 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 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-α-glucanotransferase activity, cleaving (α1 → 4)-linkages and synthesizing new (α1 → 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-α-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-α-glucanotransferase, converting amylose into a new type of α-glucan with alternating (α1 → 3)/(α 1 → 4)-linkages and with (α1 → 3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of α-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