Mutational and structural studies of the active-site residues in truncated Fibrobacter succinogenes 1,3–1,4-β-D-glucanase

1,3–1,4-β-D-Glucanases (EC 3.2.1.73) specifically hydrolyze β-1,4-glycosidic bonds located prior to β-1,3-glycosidic linkages in lichenan or β-D-glucans. It has been suggested that truncated Fibrobacter succinogenes 1,3–1,4-β-D-glucanase (TFsβ-glucanase) can accommodate five glucose rings in its active site upon enzyme–substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k_cat/K_m for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsβ-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 Å resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 Å from its position in the native enzyme complex without changing the core structure.