Fig 1.
Unrooted phylogenetic tree of representative family GH13 and GH70 protein sequences identified by BLASTp searches using the A. chroococcum GtfD 4,6-α-GTase protein as query.
Fig 2.
Predicted domain arrangement of representative members of GH70 and GH13 families with focus on GH70 4,6-α-GTase enzymes.
The crystal structures of the L. reuteri 121 GtfA glucansucrase (left) [10] and the B. licheniformis α-amylase (right) [29] are included. Domains A, B, C, IV and V are highlighted in blue, green, magenta, yellow and red, respectively. Ig2-like domains are colored in pink. The amino acid residue numbers represent the start of each domain. Conserved regions I-IV are indicated by grey rectangles. Domains A, B, C and IV were assigned in P. beijingensis GtfD by sequence comparison with L. reuteri 121 GtfB.
Table 1.
Alignment of conserved motifs I-IV of GH70 family enzymes.
(A) (putative) GtfD-like 4,6-α-GTase enzymes, (B) (putative) GtfC-like 4,6-α-GTase enzymes, (C) (putative) GtfB-like enzymes, and (D) sucrose-active enzymes. The seven strictly conserved amino acid residues in GH70 enzymes (numbered 1 to 7 above the sequences) are also conserved in GtfD-like proteins. Amino acids that constitute the catalytic triad are highlighted in bold and lightly shaded. Residues forming acceptor subsites -1, +1 and +2 in Gtf180-ΔN [7] are indicated in green, red and blue, respectively. Abbreviations at the bottom: NU = nucleophile, A/B = general acid/base, TS = transition state stabilizer. a The protein sequences are annotated by their GenBank Accession number, except for the Burkholderia sp. NFACC38-1 GtfD-like protein sequence that is labeled with its IMG/ER Gene.
Fig 3.
TLC analysis of the products synthesized by the P. beijingensis GtfD and A. chroococcum GtfD enzymes.
The P. beijingensis GtfD (A) and A. chroococcum GtfD (B) enzymes were incubated with malto-oligosaccharides (DP2-DP7), amylose V, and amylopectin at 37°C and pH 7.0 (P. beijingensis GtfD) or pH 6.5 (A. chroococcum GtfD) during 24 h. S, standard; G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose; AMV, amylose V; AMP, amylopectin; Pol, polymer.
Fig 4.
Structural analysis of the product mixtures generated after the incubation of amylose V with the P. beijingensis and A. chroococcum GtfD 4,6-α-GTase enzymes.
(A) 1H NMR spectra of the product mixtures synthesized. The spectra were recorded in D2O at 298K. Chemical shifts are shown in parts per million relative to the signal of internal acetone (δ = 2.225). Gα/β and Rα/β indicate the anomeric signals corresponding to the D-Glcp units and the reducing -(1→4)-D-Glcp units, respectively. (B) HPSEC chromatograms of the product mixtures formed by the P. beijingensis GtfD enzyme and the A. chroococcum GtfD from amylose V. The dashed line corresponds to the elution profile of the starting amylose V. The solid black and grey lines correspond to the elution profiles of products synthesized by P. beijingensis and A. chroococcum GtfD enzymes, respectively.
Fig 5.
1D 1H NMR spectrum, 2D 1H-1H TOCSY spectrum (mixing time 150 ms), and 2D 13C-1H HSQC spectrum of the HMM polysaccharide produced by the P. beijingensis GtfD enzyme from amylose.
The spectra were recorded in D2O at 298K. Peaks for (α1→4) and (α1→6) anomeric signals have been indicated. Structural reporter peaks a: H-4 for 6-substituted Glcp, b: H-4 for terminal Glcp, c: for H-4 for 4-substituted Glcp, d: H-6a for 6-substituted Glcp and e: H-6b for 6-substituted Glcp.
Table 2.
Structural characterization of the HMM and LMM polymers synthesized by the P. beijingensis GtfD enzyme from amylose V.
For comparison the characteristics of the polymer produced by the A. chroococcum GtfD enzyme are included as well.
Fig 6.
Enzymatic treatment of the P. beijingensis GtfD HMM and LMM polymers, A. chroococcum GtfD reuteran-like polymer, and L. reuteri 121 GtfB Isomalto/Malto-Polysaccharide (IMMP).
Reaction mixtures containing 5 mg ml-1 of α-glucans were incubated separately with a high dose of (A) Aspergillus oryzae α-amylase, (B) Chaetomium erraticum dextranase and (C) Klebsiella planticola pullulanase M1 for 48 h at 37ᵒC and subjected to TLC analysis. Lanes 1–4: reaction products generated by the enzymatic treatment of the P. beijingensis GtfD HMM polymer, P. beijingensis GtfD LMM polymer, reuteran-like polymer, and IMMP, respectively. Lane 5, positive controls for the α-amylase, dextranase and pullulanase digestions: amylose (A), dextran (B) and pullulan (C). Lane S, standard: glucose (G1) to maltoheptaose (G7); Pol, polymer.
Fig 7.
HPAEC profiles of the oligosaccharides formed after treatment of the P. beijingensis GtfD and A. chroococcum GtfD polymers with pullulanase M1.
The P. beijingensis GtfD HMM polymer (A), P. beijingensis GtfD LMM polymer (B), and A. chroococcum GtfD (C) were incubated with an excess of pullulanase M1 for 48 h at 37°C and pH 5. Established oligosaccharide structures are included. The identity of peaks 1–16 was assigned using commercial oligosaccharide standards and by comparison with the profile of the pullulanase hydrolysate of reuteran [17].
Fig 8.
Visual representation of composite structures for HMM and LMM P. beijingensis GtfD polymers formed from amylose V.
The composite structures contain all structural features established for the respective products. Quantities of each structural element fit with the combined data of 1D 1H NMR integration and methylation analysis, as well as enzymatic degradation studies with α-amylase, dextranase and pullulanase. For comparison the composite structure for the A. chroococcum GtfD polymer from amylose is represented as well. [15]
Fig 9.
HPAEC profiles of the oligosaccharides formed in time by the P. beijingensis GtfD and A. chroococcum GtfD enzymes from maltoheptaose.
Reaction mixtures containing 25 mM maltoheptaose were incubated with 20 μg ml-1 of P. beijingensis GtfD (A) and A. chroococcum GtfD (B) enzymes for t = 10 min, 30 min, 3 h, and 24 h, at 37°C and pH 7.0 and pH 6.5, respectively. The identity of peaks was assigned using commercial oligosaccharide standards. * Unidentified carbohydrate structures. G1, glucose; G2-G6, maltose to maltohexaose; iso-G2, isomaltose; Pa, panose.
Fig 10.
In vitro digestibility of the P. beijingensis GtfD and A. chroococcum GtfD α-glucan products in time.
Reaction mixtures containing 1 mg ml-1 of α-glucan samples were incubated with 100 U ml-1 of porcine pancreatin and rat intestinal powder extracts, concurrently, at 37°C. (A) Digestibility of the HMM and LMM polymers synthesized by the P. beijingensis GtfD and A. chroococcum GtfD enzymes from amylose V compared to the amylose V starting substrate (B) Digestibility of the gelatinized wheat starch before and after treatment with P. beijingensis GtfD and A. chroococcum GtfD enzymes. The product mixtures were obtained from 0.6% w v-1 gelatinized wheat starch by incubations with 4.6 μg ml-1 of the GtfD 4,6-α-GTase enzymes for 24 h at 37°C. The amounts of resistant (undigested after 120 min), slowly digestible (digested between 20 and 120 min) and rapidly digestible (digested within the first 20 min) carbohydrates present in the P. beijingensis GtfD- and A. chroococcum GtfD-treated wheat starches are indicated.