Aromatic amino acids in the cellulose binding domain of Penicillium crustosum endoglucanase EGL1 differentially contribute to the cellulose affinity of the enzyme.

The cellulose binding domain (CBD) of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y₄₅₁-Y₄₅₂ and Y₄₇₇-Y₄₇₈-Y₄₇₉, among which three amino acids Y₄₅₁, Y₄₇₇, and Y₄₇₈ structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y₄₅₁-Y₄₅₂ appears to contribute more to the cellulose binding than Y₄₇₇-Y₄₇₈-Y₄₇₉. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y₄₅₁, Y₄₇₇, and Y₄₇₈ which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y₄₅₁-Y₄₅₂ of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids. [ABSTRACT FROM AUTHOR]