Fungal enzymes in glycoside hydrolase family 5 subfamily 5 (GH5_5) 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, GH5_5 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 GH5_5 cellulase mutants. Their structural analysis indicated that substrate preference in fungal GH5_5 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 GH5_5 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 GH5_5 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.
Keywords: cellulase; evolution; glycoside hydrolase family 5_5; hidden Markov models; mannanase.