Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family

Abstract

Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1→6) dextrans through the specific formation of α-(1→3) linkages. To date, BRS-B is the first reported α-(1→3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1→3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.

Keywords: GH70; branching sucrase; carbohydrate; enzyme; glucansucrase; glucooligosaccharide; glycoside hydrolase; glycosylation; oligosaccharide.

SCHEME 1.

Main reactions catalyzed by GH70 sucrose-active enzymes.

FIGURE 1.

GH70 encoding genes in L. citreum NRRL B-742 genome. Yellow squares represent putative signal peptide encoding sequences. Hatched arrows indicate sequences showing >99% identity with previously characterized glucansucrases. The solid blue arrow indicates the gene previously characterized dsrB-742 (26). The green arrow stands for the brsB gene characterized in this study. Numbers below the arrows correspond to the size of the ORF in bp. The enzyme specificity was predicted according to sequence homology with other characterized GH70 enzymes.

FIGURE 2.

Schematic representation of the structural organization of BRS-B. The five different structural domains were assigned by comparison with those of GTF180-ΔN and ΔN₁₂₃-GBD-CD2: (i) domain V in red), (ii) domain IV in yellow, (iii) domain B in green, (iv) domain A in blue, and (v) domain C in purple. The catalytic residues (DED) are indicated in bold and white, YG repeats (36) with checkered red motifs and APY repeats (39) with white triangles. Signal peptide is represented by a blue circle. The red hatched regions correspond to zones for which no structural information is available.

FIGURE 3.

Analysis of the products synthesized with BRS-B enzyme from sucrose (292 mm) and dextran 1500 g·mol⁻¹ (66.6 mm). a, HPAEC-PAD chromatograms of enzymatic reaction stopped after 1 min (1) and enzymatic reaction stopped after 8 h (2). Peaks corresponding to glucose (G), fructose (F), leucrose (L), sucrose (S), and isomaltooligosaccharides with a degree of polymerization (DP) of x. b, ¹H NMR spectrum (in the anomeric region) of the products synthesized by BRS-B at 500 MHz in D₂O. Leu, leucrose; α-d-Glcp, α-d-glucose; β-d-Glcp, β-d-glucose.

FIGURE 4.

¹H NMR (a) and ¹³C NMR (b) analyses of the purified branched dextran of 2 × 10⁶ g·mol⁻¹ synthesized by BRS-B. ¹³C chemical shifts are given using acetone as reference (¹³C = 31.08 ppm).

FIGURE 5.

Effect of temperature (a), pH (b), and the addition of divalent ions (c) on BRS-B-Δ1 transferase activity. Results are given as the means ± S.D. n = 3.

FIGURE 6.

Synthesis of α-(1→3)-branched dextrans using BRS-B-Δ1 and varying sucrose:acceptor initial ratio (mass concentrations). a, determination of the percentage of glucosyl moieties from sucrose incorporated into free glucose (black), leucrose (light gray), and α-(1→3) dextran (dark gray). The production of other oligosaccharides was negligible. b, control of the amount of α-(1→3) linkages in branched dextrans of 1500 (▴) and 2 × 10⁶ g·mol⁻¹ (○) as the function of the sucrose:acceptor initial ratio.

FIGURE 7.

Surface representation of BRS-B model and ΔN₁₂₃-GBD-CD2 crystal structure (PDB code 3TTQ). Domains A, B, C, IV, and V are colored in light blue, green, pink, yellow, and red, respectively. Variable loop regions are shown in bright colors. Sucrose is shown in the active site as yellow and red sticks for carbon and oxygen atoms, respectively.

FIGURE 8.

Comparison of the active sites of BRS-B, ΔN₁₂₃-GBD-CD2 (PDB code 3TTQ) and GTF-180-ΔN glucansucrase (PDB code 3HZ3). a, superimposition of BRS-B with GTF-180-ΔN in complex with sucrose (PDB code 3HZ3). Side chains forming the subsites −1 and +1 are shown as sticks. Sucrose is depicted as yellow and red sticks. GTF-180-ΔN is represented in gray, whereas domains A and B of BRS-B are represented in blue and green, respectively. b, superimposition of BRS-B with ΔN₁₂₃-GBD-CD2 structure (PDB code 3TTQ). ΔN₁₂₃-GBD-CD2 is represented in gray. Domain A and domain B of BRS-B are represented in blue and green, respectively.

FIGURE 9.

Analysis of the products synthesized using BRS-C and BRS-D enzymes from sucrose (292 mm) and dextran 1500 g·mol⁻¹ (66.6 mm). a, comparison of BRS-B and BRS-C HPAEC-PAD chromatograms. (1) Enzymatic reaction at the initial time (t = 0 min), (2) enzymatic reaction using BRS-B stopped after 8 h, and (3) enzymatic reaction using BRS-C stopped after 8 h. nC, nanocoulombs. b, comparison of BRS-A and BRS-D HPAEC-PAD chromatograms. (1) Enzymatic reaction at the initial time (t = 0 min), (2) enzymatic reaction using BRS-B stopped after 8 h, and (3) enzymatic reaction using BRS-C stopped after 8 h. G, peak corresponding to glucose fructose (F), leucrose (L), sucrose (S), and isomaltooligosaccharides with a degree of polymerization (DP) of x. c, ¹H NMR spectrum of the products synthesized by BRS-B (1) and BRS-C (2) at 500 MHz in D₂O leucrose (Leu), α-d-glucose (α-d-Glcp), β-d-glucose (β-d-Glcp), α-(1→6)-linked glucosyl residues (α-(1→6)), and α-(1→3)-linked glucosyl residues (α-(1→3)). d, ¹H NMR spectrum of the products synthesized by BRS-A (1) and BRS-D (2) at 500 MHz in D₂O. The ¹H NMR spectra of the BRS-A modified dextran displayed all the chemical shift characteristics of α-(1→2)-branched dextran. The anomeric region of the spectrum contained three main resonances corresponding, respectively, to anomeric resonance of α-(1→6)-linked d-Glcp residues of the main linear chain with free carbon 2 (4.98 ppm, peak C), to anomeric resonance from α-(1→6)-linked d-Glcp residues of the main linear chain, with the carbon 2 involved in an α-(1→2) linkage with a branched glucosyl unit (5.18 ppm, peak A), to anomeric resonance from α-(1→2)-linked d-Glcp (5.11 ppm, branching points, peak B).

FIGURE 10.

Phylogenetic tree of GH70 enzymes. Each enzyme is labeled with its GenBank^TM accession number and its origin. The species of microorganism are indicated with Ln. for Leuconostoc, Lb. for Lactobacillus, S. for Streptococcus, and W. for Weissella. Glucansucrase enzymes are highlighted in green, α-4,6 glucanotransferases are in pink, and branching sucrases are in bold and purple. The scale bar corresponds to a genetic distance of 0.05 substitutions per position.