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.