Structural Characterization of Bioengineered α-d-Glucans Produced by Mutant Glucansucrase GTF180 Enzymes of Lactobacillus reuteri Strain 180

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

doi:10.1021/bm801240r

Cited by 52 publications

(68 citation statements)

References 28 publications

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“…In GtfC homologs, the conserved residue W1065 (Gtf180 L. reuteri 180 numbering) is replaced by a Tyr, as is the case in GtfB-like 4,6-␣-glucanotransferases. Moreover, the amino acid residues at positions 1137, 1140, and 1141, known to be important for glucosidic linkage specificity in glucansucrases (28)(29)(30), are Gln, Lys, and Asn, respectively, in GtfC homologs, similar to GtfB-like 4,6-␣-glucanotransferases. Apart from the well-recognized I-to-IV homology regions, the three additional conserved regions (VI, V, and VII) proposed to be related to the maintenance of the structure and to a given enzyme specificity in GH13 family proteins (6,31,32) were identified in the GH70 and GH13 sequences.…”

Section: Resultsmentioning

confidence: 99%

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Gangoiti

Pijning

Dijkhuizen

2016

Appl Environ Microbiol

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105

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The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing ␣-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-␣-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 ␣1¡4 linkages, and synthesize linear ␣1¡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 ␣1¡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-␣-glucanotransferase activity with maltooligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB-and GtfCtype 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 ␣-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 (␤/␣) 8 barrel. The GtfC 4,6-␣-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between ␣-amylase and glucansucrase enzymes.T he starch-and sucrose-acting enzymes of the glycoside hydrolase 13 (GH13) and GH70 families are evolutionarily related, displaying similar protein folds and activity mechanisms (retaining, covalent intermediate, double displacement mechanism, three catalytic residues), constituting clan GH-H (http://www .cazy.org) (B. Svensson and Š. Janeček, glycoside hydrolase family 13 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015; M. Remaud-Simeon, glycoside hydrolase family 70 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015). They differ in their overall activities, degrading or modifying ␣-glucan substrates (starch, maltodextrins, GH13) (1, 2) or synthesizing ␣-glucan products (from sucrose, GH70) (3), e.g., by the Lactobacillus reuteri 121 GtfA enzyme (4). GH13 proteins have three domains (A, B, and C) with a common catalytic (␤/␣) 8 fold (triosephosphate isomerase [TIM] barrel); their active site is located in an open cavity between the A and B domains (5, 6). GH70 proteins share this domain organization, but their (␤/ ␣) 8 barrel is circularly permuted. Moreover, they possess unique domains IV and V (7). Recently, we ha...

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Section: Resultsmentioning

confidence: 99%

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Gangoiti

Pijning

Dijkhuizen

2016

Appl Environ Microbiol

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105

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“…LB agar plates were made by adding 1.5% agar to the LB medium. The plasmid p15GTF180-⌬N-SX, containing N-terminally truncated GTF180 (residue 742-1772), was used for mutagenesis and protein production (27).…”

Section: Methodsmentioning

confidence: 99%

“…The relative intensity of the H-4 signal (t4, between ϳ␦ 3.40 and 3.45) stemming from terminal residues of ␣-glucan polysaccharides is an indicator for the amount of branching (Fig. 3b) (27,29,39). Examination of NMR spectra of polysaccharides produced by Ala-978 mutants with a larger side chain (A978L, A978P, A978F, and A978Y) revealed that the intensity of the t4 signal was reduced, indicating a decreased amount of branched linkages; those of Ala-978 mutations with a small amino acid resi-due (A978G and A978S) did not show such a change (Fig.…”

Section: Ala-978 Has An Important Role In Branched Linkage Formation-mentioning

confidence: 99%

“…Second, the crystal structure of GTF180-⌬N in complex with maltose revealed this acceptor substrate bound in subsites ϩ1 and ϩ2. At subsite ϩ2, the residues following the transition state stabilizer (Asp-1136) in homology region IV, which have been shown to be important for linkage specificity in previous mutagenesis studies (9,22,(25)(26)(27)(28), are located close to the reducing end moiety of maltose, especially Ser-1137, which has a direct hydrogen bond with the ϩ2 C1 hydroxyl group (12). These observations confirmed the involvement of these residues in forming acceptor binding sites as predicted in previous studies and explain the altered linkage specificity caused by mutating these residues.…”

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confidence: 99%

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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 Characterization of Bioengineered α-d-Glucans Produced by Mutant Glucansucrase GTF180 Enzymes of Lactobacillus reuteri Strain 180

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Cited by 52 publications

References 28 publications

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

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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