Deciphering functional diversity and structural determinants of substrate specificity in fungal glycoside hydrolase family 5₅ cellulases

Fungal enzymes in glycoside hydrolase family 5 subfamily 5 (GH5₅) display notable catalytic diversity, efficiently degrading cellulose and sometimes mannan. However, the structural determinants and molecular mechanisms governing substrate preference in this enzyme family remain unclear. In this study, GH5₅ enzymes from fungi were systematically classified using profile-based sequence models and functionally characterized. Saturation mutagenesis combined with high-resolution crystal structure analysis of the bifunctional enzyme BsCel5B, exhibiting cellulase (CEL) activity of 941 ± 17 U/mg and mannanase (MAN) activity of 1, 736 ± 34 U/mg, was employed to identify key residues controlling substrate specificity. Residue T100 in BsCel5B was identified as a major structural contributor associated with significant shifts in substrate preference. The T100V and T100N mutations resulted in 2. 0-fold increases in MAN activity and 2. 5-fold increases in CEL activity, respectively, generating bifunctional enzymes with enhanced substrate-specific activities. Similar substrate specificity trends were observed in several GH5₅ cellulase mutants. Their structural analysis indicated that substrate preference in fungal GH5₅ enzymes might be shaped by residual network-mediated alterations of the active-site geometry, with T100 acting as a second-shell regulatory element within a cooperative residue network. Together, these findings suggest a mechanistic framework for engineering catalytic specificity in GH5₅ enzymes. IMPORTANCECellulose and mannan are major components of plant biomass, and enzymes capable of efficiently breaking them down are essential for sustainable biofuel production and biomass utilization. Fungal enzymes in GH5₅ are widely used for these purposes, yet their functional diversity has been difficult to predict or control. Substrate preference in these enzymes can be modulated by altering a single amino acid, offering a promising approach for tuning enzyme activity. The identification of a key residue that influences the balance between cellulose and mannan degradation provides valuable insights for engineering enzymes with tailored functions. These findings contribute to a deeper understanding of fungal biomass-degrading enzymes and support the rational design of more efficient catalysts for industrial and environmental applications.