Essential catalytic role of Glu134 in endo‐β‐1,3‐1,4‐d‐glucan 4‐glucanohydrolase from B. licheniformis as determined by site‐directed mutagenesis

Abstract

Site‐directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo‐β‐1,3‐1,4‐d‐glucan 4‐glucanohydrolase (β‐glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinan: E. coli and their kinetics analysed with barley β‐glucan. Replacement of Glu¹³⁴ by Gin produced a mutant (E134Q) that retains less than 0.3% of the wild‐type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild‐type indicative of their participation in substrate binding and transition‐state complex stabilization. Glu¹³⁴ is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme‐like mechanism for this family of Bacillus β‐glucan hydrolases with Glu¹³⁴ being the essential acid catalyst.