Catalytic Mechanism of a Novel Glycoside Hydrolase Family 16 "Elongating" β-Transglycosylase

Abstract

Carbohydrates are complex macromolecules in biological metabolism. Enzymatic synthesis of carbohydrates is recognized as a powerful tool to overcome the problems associated with large scale synthesis of carbohydrates. Novel enzymes with significant transglycosylation ability are still in great demand in glycobiology studies. Here we report a novel glycoside hydrolase family 16 "elongating" β-transglycosylase from Paecilomyces thermophila (PtBgt16A), which efficiently catalyzes the synthesis of higher polymeric oligosaccharides using β-1,3/1,4-oligosaccharides as donor/acceptor substrates. Further structural information reveals that PtBgt16A has a binding pocket around the -1 subsite. The catalytic mechanism of PtBgt16A is partly similar to an exo-glycoside hydrolase, which cleaves the substrate from the non-reducing end one by one. However, PtBgt16A releases the reducing end product and uses the remainder glucosyl as a transglycosylation donor. This catalytic mechanism has similarity with the catalytic mode of amylosucrase, which catalyzes the transglycosylation products gradually extend by one glucose unit. PtBgt16A thus has the potential to be a tool enzyme for the enzymatic synthesis of new β-oligosaccharides and glycoconjugates.

Keywords: enzyme catalysis; enzyme mechanism; enzyme structure; glycoside hydrolase; protein crystallization.

FIGURE 1.

Sequence analysis of PtBgt16A. A, domain analysis of PtBgt16A-full full-length protein. B, structural sequence alignment of some GH family 16 members. Identical residues are shown in white on a red background, and conservative residues are shown in red on a white background. Two catalytic glutamic acid residues, Glu¹¹⁷ and Glu¹²², are marked by red dots. The key residue, Trp¹¹², of PtBgt16A is marked by a red star. Four disulfide bonds of PtBgt16A are marked by green numbers underneath the relative residues. The sequences of PtBgt16A, P. thermophila β-1,3-1,4-glucanase (PtLic16A; Protein Data Bank code 3WDT), P. chrysosporium β-1,3(4)-glucanase (PcLam16A; Protein Data Bank code 2CL2), Zobellia galactanivorans β-1,3-glucanase (ZgLamCGH16; Protein Data Bank code 4CRQ), Rhodothermus marinus β-1,3-glucanase (RmLamR; Protein Data Bank code 3ILN), and Nocardiopsis sp. β-1,3-glucanase (BglF; Protein Data Bank code 2HYK) were aligned using T-Coffee (43), and the figure was produced using ESPript (44).

FIGURE 2.

SDS-PAGE of proteins during purification of the recombinant PtBgt16A by nickel-iminodiacetic acid. Lane M, standard protein molecular weight markers; lane 1, supernatant of lysate cells; lane 2, purified enzyme.

FIGURE 3.

Catalytic ability of PtBgt16A. TLC analysis of products hydrolyzed by PtBgt16A toward laminarioligosaccharides (A) and cello-oligosaccharides (B) is shown. M, marker; G, glucose; L2–L6, laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose, respectively; C2–C5, cellobiose, cellotriose, cellotetraose, and cellopentose, respectively. Left, before reaction; right, after reaction. MALDI-TOF MS analysis of transglycosylation reaction products by PtBgt16A toward laminaritriose (C) and cellotriose (D) is shown. The peaks in the spectra correspond to the monoisotopic masses of sodium adducts [M + Na]⁺ of the oligosaccharides. a.u., arbitrary units.

FIGURE 4.

TLC analysis of transglycosylation reaction course by PtBgt16A. Purified PtBgt16A (1 unit/ml) was added to 1% (w/v) laminarioligosaccharides in 50 mm sodium acetate buffer, pH 5.5, and then incubated at 50 °C for 2 h. M, marker sugars; G1, glucose; L2–L6, laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose, respectively.

FIGURE 5.

The effects of temperature and pH on activity and stability of the purified PtBgt16A. A, optimal pH. B, pH stability. C, optimal temperature. D, thermostability. The optimal pH was determined in different buffers, including McIlvaine buffer (■), sodium acetate buffer (●), Tris-HCl buffer (▴), and glycine-NaOH buffer (▾). Laminaritriose was used as substrate to determine the enzymatic characterization of PtBgt16A.

FIGURE 6.

Two-dimensional NMR data (HMBC) of transglycosylation products by PtBgt16A. A, the enlarged picture of HMBC spectra of cellotriose before (1) and after (2) reaction. B, the enlarged picture of HMBC spectra of laminaritriose before (1) and after (2) reaction. Characteristic chemical shifts occurred at 84 and 78 ppm, indicating a β-1,3 and β-1,4 linkage in the transglycosylation products, respectively. Transglycosylation reaction (30 ml) was performed with PtBgt16A (1 unit/ml) and 1% (w/v) laminaritriose or cellotriose in 50 mm sodium acetate buffer, pH 5.5, at 50 °C for 2 h.

FIGURE 7.

Crystal structure of PtBgt16A. A, overall structure and the unique catalytic groove of PtBgt16A. The two PtBgt16A molecules from the dimer are shown, one as a ribbon diagram and the other by electrostatic potential surface. The unique loop is highlighted in green. The electron densities of the unique loop are shown as the σA-weighted mF_o − DF_c omit map contoured at the 3.0 σ level in an amplified view. B, substrate interactions of PtBgt16A. Left, superposition of PtBgt16A on other GH family 16 enzymes is shown as a ribbon diagram with ribbons colored according to each enzyme: PtBgt16A in green, PtLic16A (Protein Data Bank code 3WDT) in blue, PcLam16A (Protein Data Bank code 2W39) in purple, and PcLam16A (Protein Data Bank code 2WLQ) in yellow. The binding pocket of PtBgt16A is shown in surface representation. Right, substrate interactions of PtBgt16A by ligands superposed. The −1 site glucose residue is from PcLam16A-laminaribiose complex (Protein Data Bank code 2W39), and the +1 and +2 site glucose residues are from PcLam16A-laminariheptaose complex (Protein Data Bank code 2WLQ). All of the superposed ligands are shown in stick representation and colored in yellow. The amino residues from PtBgt16A, PtLic16A, and PcLam16A are colored green, blue, and purple, respectively.

FIGURE 8.

Catalytic mechanism of PtBgt16A transglycosylation. A, schematic representation of substrate interactions of PtBgt16A. The oligosaccharide is drawn as sticks. The hydrogen bonding interactions are shown as dotted lines. B, schematic mechanism of the catalysis reaction by PtBgt16A. C, enzyme activity of PtBgt16A mutants. Data represent the mean ± S.D. of three independent experiments (n = 3).

FIGURE 9.

B-factor putty (A) and sequence alignment (B) of the unique loop of PtBgt16A. The key residue, Tyr¹¹², of PtBgt16A is marked by a red star. The sequences of PtBgt16A (Protein Data Bank code 5JVV), PtLic16A (Protein Data Bank code 3WDT), PcLam16A (Protein Data Bank code 2CL2), ZgLamCGH16 (Protein Data Bank code 4CRQ), RmLamR (Protein Data Bank code 3ILN), and BglF (Protein Data Bank code 2HYK) were aligned using T-Coffee (43), and the figure was produced in ESPript (44).