Structural Analysis of Bioengineered α-<scp>d</scp>-Glucan Produced by a Triple Mutant of the Glucansucrase GTF180 Enzyme from Lactobacillus reuteri Strain 180: Generation of (α1→4) Linkages in a Native (1→3)(1→6)-α-<scp>d</scp>-Glucan

Leeuwen, Sander S. van; Kralj, Slavko; Gerwig, Gerrit J.; Dijkhuizen, Lubbert; Kamerling, Johannis P.

doi:10.1021/bm800410w

Cited by 33 publications

(55 citation statements)

References 26 publications

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“…The 1D 1 H NMR spectrum (Fig. 5a) showed α-anomeric signals at δ 5.41 and 5.37; this region may contain anomeric signals of both (α1 → 4) and (α1 → 3) linked α- D -Glc p residues48. Therefore, fractions F1-F5 were also subjected to methylation analysis in order to determine the presence of (α1 → 4) and (α1 → 3) linkages and to determine the degree of branching.…”

Section: Resultsmentioning

confidence: 99%

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

Gangoiti

Leeuwen

Gerwig

et al. 2017

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 99%

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

Gangoiti

Leeuwen

Gerwig

et al. 2017

Sci Rep

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“…1) (9, 22, 25, 27). GTF180 contains SNA at this position; mutations targeting these residues altered linkage specificity of GTF180 and even introduced a third type of linkage ((␣134) linkage) not present in the ␣-glucan produced by wild-type enzyme (27,29,40). The corresponding tripeptide in the dextransucrase DSRS (SEV) has been the subject of mutation studies (22,31).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180

Meng

Pijning

Dobruchowska

et al. 2015

Journal of Biological Chemistry

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␣-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-⌬N from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite ؉1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (␣136) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the ؉1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structurefunction relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailormade ␣-glucans.

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“…Glc(a1 / 6) Glc(a1 / 3)Glc(a1 / 6)Glc [28]. Taking into account composite models of a-D-glucans generated from sucrose using wild-type and mutant Gtf180-DN glucansucrases as biocatalyst [28,30,31], the structure of one of the expected minor products is hypothesized to be an elongation of RebA-G2a with a Glc(a1 / 6) residue [Glc7(a1 / 6)Glc6(a1 / 6)Glc5(a1 / 6)Glc1(b1 / ].…”

Section: Structural Analysis Of Hplc Fraction F4mentioning

confidence: 99%

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

Gerwig

Poele

Dijkhuizen

et al. 2017

Carbohydrate Research

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Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation Gerwig, Gerrit; te Poele, Evelien; Dijkhuizen, Lubbert; Kamerling, Johannis P a b s t r a c tThe wild-type Gtf180-DN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the a-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 a-D-glucosylated at the steviol C-19 b-D-glucosyl moiety (55% conversion). The main product is a mono-(a1 / 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 (a1 / 3)-and (a1 / 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. IntroductionThe leaves of the Stevia rebaudiana BERTONI plant contain a high variety of sweet substances, called steviol glycosides [1,2], and so far more than 40 different structures have been elucidated (see review Ref. [3]). Stevioside (~5e20% w/w of dried leaves) and rebaudioside A (~2e5% w/w of dried leaves) are the most abundant components (Fig. 1), tasting about 200e300 times sweeter than sucrose (0.4% aqueous solution). Structurally, steviol glycosides have ent-13-hydroxykaur-16-en-19-oic acid as aglycone, called steviol, but are differing in carbohydrate composition at the C-13-tert-hydroxyl and C-19-carboxyl functions.Due to the growing awareness and concerns for human health related to excessive consumption of sugar (sucrose), the application of steviol glycosides as non-caloric bio-alternatives for sucrose and as substitutes for artificial (synthetic) sweeteners is strongly promoted nowadays [4e8]. Since a couple of years, steviol glycosides have been permitted for use as food additive and sweetener in the USA [9] and in Europe (E960) [10,11]. However, despite their intense sweetness and diverse beneficial pharmacological properties [12e15], the main drawback for successful commercialization of Stevia sweeteners is their slight bitterness and unpleasant (metallic) aftertaste, experienced by more than half of the human population.For natural steviol glycosides with b-D-glucopyranosyl units as constituents, it has been reported that the ratio of the number of glucose units at the C-13 site to that at the C-19 site of the steviol core has a relationship with the sweetness as well as with the quality of taste of the steviol glycosides [16,17]. To improve the Abbreviations: FID, free induction decay; FWHM, full width at half maximum; GLC-EI-MS, gas-liquid chromatography electron impact mass spectrometry; HPAEC, high-pH anion-exchange chromatography; HSQC, 1 H-detected hetero-nucl...

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Structural Analysis of Bioengineered α-d-Glucan Produced by a Triple Mutant of the Glucansucrase GTF180 Enzyme from Lactobacillus reuteri Strain 180: Generation of (α1→4) Linkages in a Native (1→3)(1→6)-α-d-Glucan

Cited by 33 publications

References 26 publications

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

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

Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180

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

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