Unveiling the Structural and Biochemical Characteristics of an Acidophilic β-Mannanase from Soybean (Glycine max)

Abstract

Endo-1,4-β-mannanase (EC 3.2.1.78) cleaves β-1,4-mannans in cell walls, facilitating endosperm softening and seed germination. Here, we present the structural and biochemical characterization of GmMAN19-1, a GH5_7 β-mannanase from soybean (Glycine max), a crop with substantial agricultural importance. GmMAN19-1 is specifically expressed in cotyledons during postgermination and exhibits acidophilic activity optimal at pH 4.6 and 40 °C. Crystal structures of GmMAN19-1 were determined at 1.39 and 2.62 Å in the apo form and in complex with mannopentaose (M5), respectively. The structure adopts a canonical (α/β)₈ TIM barrel fold with a V-shaped active site groove. Notably, two distinct M5 binding modes were identified, suggesting dual functionality involving hydrolytic and transglycosylation activities. Site-directed mutagenesis further validated key catalytic and substrate-interacting residues: E186A abolished enzymatic activity, while Q267W altered transglycosylation product profiles and enhanced activity toward branched substrates. The binding groove can accommodate galactose side chains, supporting partial activity toward galactomannans. These findings provide comprehensive insights into the substrate specificity and catalytic mechanism of plant β-mannanases and establish GmMAN19-1 as a potential candidate for applications in food processing, biomass conversion, and industrial biotechnology, particularly due to its acidophilic nature and enhanced activity toward branched mannans.

Keywords: GmMAN19-1; X-ray crystallography; branched mannans; soybean; β-mannanase.

1

pH dependence and stability of GmMAN19-1. (A) The optimal pH for GmMAN19-1 activity was determined by measuring enzyme activity across a pH range of 2.0–10.6. (B) Circular dichroism (CD) spectra of GmMAN19-1 at different pH values. (C) pH stability of GmMAN19-1 was assessed by preincubating the enzyme in buffers of varying pH at 4 °C for 16 h, followed by activity measurement. (D) CD spectra of GmMAN19-1 after 16 h of incubation at different pH values. β-Mannanase activity was measured using the DNS method with LBG as the substrate.

2

Temperature dependence and stability of GmMAN19-1. (A) The optimal temperature for GmMAN19-1 activity was determined by measuring enzyme activity at temperatures ranging from 25 to 50 °C. (B) Thermal stability of GmMAN19-1 was assessed by preincubating the enzyme at various temperatures for different durations before measuring residual activity. (C) Thermal denaturation of GmMAN19-1 was analyzed using circular dichroism (CD) spectroscopy at 222 nm in 25 mM phosphate buffer (pH 7.5). β-Mannanase activity was measured using the DNS method with LBG as the substrate.

3

Overall structure of GmMAN19-1. (A) Cartoon representation of the GmMAN19-1 overall structure with α-helix, β-strand, and loop colored as cyan, yellow, and purple, respectively. (B) Topology diagrams use the same color scheme as the cartoon representations.

4

Structure of GmMAN19-1 in complex with mannopentaose (M5). (A) The GmMAN19-1/M5 complex structure reveals two M5 molecules bound within the active-site region. Mannopentaose is depicted as sticks, with carbon atoms in yellow, and the 2Fo-Fc electron density map is contoured at 1.0 σ. (B) Surface representation of GmMAN19-1, highlighting the mannopentaose (yellow carbons) positioned within the active-site cleft (green surface). (C) Close-up view of the bound mannopentaose, illustrating two distinct binding modes. The 2Fo-Fc electron density map is contoured at 1.0 σ. (D) Interactions of the substrate in the active site of GmMAN19-1/M5. Green residues form hydrogen bonds with mannopentaose, whereas magenta residues interact with the ligand via hydrophobic and van der Waals forces. Cyan residues represent catalytic residues (E186 and E304). Mannopentaose molecules are shown as sticks, with carbon atoms in yellow and orange, while water molecules are depicted as blue spheres.

5

Michaelis–Menten plots of GmMAN19-1 using locust bean gum (LBG), guar gum (GG), or ivory nut mannan (INM) as the substrate. (A) Plot of GmMAN19-1 using LBG as a substrate; the measurements were performed at 40 °C for 5 min. (B) Plot of GmMAN19-1 using LBG as a substrate; the measurements were performed at 30 °C for 10 min. (C) Plot of GmMAN19-1 using GG as a substrate; the measurements were performed at 40 °C for 10 min. (D) Plot of GmMAN19-1 using GG as a substrate, the measurements were performed at 30 °C for 15 min. (E) Plot of GmMAN19-1 using INM as a substrate, the measurements were performed at 40 °C for 10 min. (F) Plot of GmMAN19-1 using GG as a substrate, the measurements were performed at 30 °C for 10 min.