Characterization of two 1,3-β-glucan-modifying enzymes from Penicillium sumatraense reveals new insights into 1,3-β-glucan metabolism of fungal saprotrophs

Abstract

Background: 1,3-β-glucan is a polysaccharide widely distributed in the cell wall of several phylogenetically distant organisms, such as bacteria, fungi, plants and microalgae. The presence of highly active 1,3-β-glucanases in fungi evokes the biological question on how these organisms can efficiently metabolize exogenous sources of 1,3-β-glucan without incurring in autolysis.

Results: To elucidate the molecular mechanisms at the basis of 1,3-β-glucan metabolism in fungal saprotrophs, the putative exo-1,3-β-glucanase G9376 and a truncated form of the putative glucan endo-1,3-β-glucosidase (ΔG7048) from Penicillium sumatraense AQ67100 were heterologously expressed in Pichia pastoris and characterized both in terms of activity and structure. G9376 efficiently converted laminarin and 1,3-β-glucan oligomers into glucose by acting as an exo-glycosidase, whereas G7048 displayed a 1,3-β-transglucanase/branching activity toward 1,3-β-glucan oligomers with a degree of polymerization higher than 5, making these oligomers more recalcitrant to the hydrolysis acted by exo-1,3-β-glucanase G9376. The X-ray crystallographic structure of the catalytic domain of G7048, solved at 1.9 Å of resolution, consists of a (β/α)₈ TIM-barrel fold characteristic of all the GH17 family members. The catalytic site is in a V-shaped cleft containing the two conserved catalytic glutamic residues. Molecular features compatible with the activity of G7048 as 1,3-β-transglucanase are discussed.

Conclusions: The antagonizing activity between ΔG7048 and G9376 indicates how opportunistic fungi belonging to Penicillium genus can feed on substrates similar for composition and structure to their own cell wall without incurring in a self-deleterious autohydrolysis.

Keywords: 1,3-β-Glucan metabolism; 1,3-β-Transglucanase; Cell wall-modifying enzymes; Exo-1,3-β-glucanase; Penicillium; TIM-barrel.

Fig. 1

Enzymatic characterization of exo-1,3-β-glucanase G9376. (a) pH- and (b) temperature-dependent activity of exo-1,3-β-glucanase G9376 toward 0.2% (w/v) laminarin as determined by the reducing sugar assay. (c) Residual activity of exo-1,3-β-glucanase G9376 toward 0.2% (w/v) laminarin after 1 h-incubation at different temperatures as determined by the reducing sugar assay. (d) Specific activity expressed as Units (µmol reducing end/min, µmol Glc/min) per mg of exo-1,3-β-glucanase G9376 toward LAM5ol. The amounts of reducing ends and Glc were determined by the reducing sugar and GO-POD assays, respectively. Values are mean ± SD, n = 3. Analyses shown in (b–d) were performed at pH 5.0. [LAM5ol, 1,3-β-D-laminaripentaitol borohydride; Glc, D-Glucose]

Fig. 2

Analysis of degradation products obtained from different 1,3-β-glucan oligomers upon incubation with exo-1,3-β-glucanase G9376 and the enzyme ΔG7048. Chromatographic analysis of equal amounts of five different 1,3-β-glucan oligomers (upper panel) alone, (middle panel) upon 1 h incubation with exo-1,3-β-glucanase G9376 and (lower panel) upon 24 h incubation with the enzyme ΔG7048. In the upper panel, glucose was also analyzed. [Glc D-Glucose, LAM2 laminaribiose, LAM3 laminaritriose, LAM4, laminaritetraose, LAM5 laminaripentaose, LAM6 laminarihexaose]

Fig. 3

Analysis of degradation products obtained from a ΔG7048-pretreated LAM5 and laminarin upon incubation with exo-1,3-β-glucanase G9376. Chromatographic analysis of (upper panel) glucose, five different 1,3-β-glucan oligomers and laminarin, and of (lower panel) degradation products obtained from a ΔG7048-pretreated LAM5 upon 1 h-incubation with exo-1,3-β-glucanase G9376 (ΔG7048-pretreated LAM5 + G9376), and from laminarin upon 1 h- (Laminarin + G9376) and 24 h-incubation with exo-1,3-β-glucanase G9376 [Laminarin + G9376 (24 h)]. ΔG7048-pretreated LAM5 was analyzed as control. Black arrow indicates the peak corresponding to the degradation by-product X. [Glc D-Glucose, LAM2 laminaribiose, LAM3 laminaritriose, LAM4 laminaritetraose, LAM5 laminaripentaose, LAM6 laminarihexaose]

Fig. 4

Scheme of the reaction catalyzed by 1,3-β-transglucanase ΔG7048 using LAM5 as a substrate, and degradation of the product(s) by exo-1,3-β-glucanase G9376. Top panel: hydrolysis catalyzed by exo-1,3-β-glucanase G9376 on LAM5 as deduced from the analyses shown in Figs. 1d, 2 and Additional File 2: Fig. S3. Bottom panel: transglycosylation catalyzed by 1,3-β-transglucanase ΔG7048 using LAM5 as substrate, and subsequent hydrolysis of the hybrid octamer(s) by exo-1,3-β-glucanase G9376. Red line indicates the putative 1,6-β-glycosidic linkage. The molar ratio of degradation products obtained from both enzymatic reactions (2xLAM5 + G9376) and (2xLAM5 + ΔG7048 + G9376) are reported on the right. The reducing end (R) of each LAM5 oligomer is also indicated [Glc D-Glucose, LAM2 laminaribiose, LAM3 laminaritriose, LAM5 laminaripentaose; Y, residue n. 6 or n. 7 or n. 8]

Fig. 5.

3D-structure of 1,3-β-transglucanase ΔG7048. a Rainbow colored cartoon of the structure of ΔG7048 with the catalytic residues in stick representation. b Structural superposition of ΔG7048 (blue) with the endo-1,3-β-glucanase of S. tuberosum (PDB: 3ur8, yellow)

Fig. 6

Comparison between ΔG7048 and RmBgt17A. a Structural-based sequence alignment between ΔG7048 and RmBgt17A. Identical residues are shown on a red background and conservatively mutated residues are shown in red on white background. The catalytic residues, E111 and E222, are marked with blue dots. Residues reported to be involved in direct or H₂O-mediated interaction with the substrate in RmBgt17A are underlined in green and orange, respectively. Residues limiting the access to the catalytic cleft in RmBgt17A are underlined in black. Regions corresponding to the 4 sites described in panel b are indicated by black boxes. The sequences were aligned by Chimera and the figure was produced in ESPript. b Structural superposition of ΔG7048 (blue) with the ligand free form of RmBgt17A (PDB: 4wtp, pink). c structural superposition of ΔG7048 (blue) with (i) ligand free RmBgt17A (PDB: 4wtp, pink), (ii) LAM2-RmBgt17A (PDB: 4wtr, green) and (iii) LAM3-RmBgt17A (PDB: 4wts, yellow). d Surface representation of ΔG7048 (left) and RmBgt17A (PDB: 4wtp, right) with substrates from RmBgt17A structures (PDB: 4wtr, PDB: 4wts) located in the catalytic cleft without further docking experiment or energy minimization procedure. e Close view of the substrate binding region of structures superposed in c. Catalytic residues and those directly contacting the substrates in RmBgt17A with the corresponding ones in ΔG7048 are representing in sticks (numbering according to the ΔG7048 sequence)