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

ABSTRACT: 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.

SUBMITTER: Pijning T

PROVIDER: S-EPMC8587608 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Pijning Tjaard T Gangoiti Joana J Te Poele Evelien M EM Börner Tim T Dijkhuizen Lubbert L

Journal of agricultural and food chemistry 20211028 44

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 ...[more]

PMID: 34708648

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Project description:BackgroundThe GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme.MethodsDenaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC.ResultsExpression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB.ConclusionIn view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Project description:Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

Dataset Information

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

Publications

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

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