FEBS Letters | |
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 | |
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
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