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

FEBS Letters

Lloberas, J.¹  Juncosa, M.¹  Querol, E.¹  Planas, A.¹ 

[1] Institut de Biologia Fonamental V. Villar Palasi and Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

关键词: Site-directed mutagenesis;    Active site;    β-1;    3-1;    4-Glucanase;    B. licheniformis;    gluL;    gene coding for β-glucanase from B. licheniformis;    D;    aspartic acid;    DNS;    dinitrosalicilic acid;    E;    glutamic acid;    LB and 2YT;    bacterial media as described in Sambrook et al. [28];    N;    asparagine;    PAGE;    polyacrylamide gel electrophoresis;    Q;    glutamine;    SDS;    sodium dodecyl sulfate;    wt;    wild-type;   

DOI  :  10.1016/0014-5793(92)81262-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown