科研之友
微信
新浪微博
Facebook
分享链接
摘要
Glycosynthases from more than 16 glycosidase families have been developed for the efficient synthesis of oligosaccharides and glycoconjugates. beta-1,3-1,4-Glucan oligo- and polysaccharides with defined sequences can be quantitatively achieved with the glycosynthases derived from Bacillus licheniformis beta-1,3-1,4-glucanase. The screening of a nucleophile saturation library of this enzyme yielded the unexpected E134D mutant which has high glycosynthase efficiency (25% higher k(cat) than the best glycosynthase to date, E134S) but also retains some hydrolase activity (2% relative to the wild-type enzyme). Here, we report the biochemical and structural analyses of this mutant compared to E134S and wild-type enzymes. E134D shows a pH profile of general base catalysis for the glycosynthase activity, with a kinetic pK(a) (on k(cat)/K-M) assigned to Glu138 of 5.8, whereas the same residue acts as a general acid in the hydrolase activity with the same pK(a) value. The pK(a) of Glu138 in the wt enzyme was 7.0, a high value due to the presence of the catalytic nucleophile Glu134 which destabilizes the conjugate base of Glu138. Thus, the pK(a) of Glu138 drops 1.1 pH units in the mutant relative to the wild-type enzyme meaning that the larger distance between carboxylates in positions 138 and 134 (5.6 angstrom for wt, 7.0 angstrom for E134D) and/or a new hydrogen bonding interaction with a third Asp residue (Asp136) in the mutant reduces the effect of the negatively charged Asp134. In consequence, the pK(a) of Glu138 has a similar pK(a) value in the E134D mutant than in the other glycosynthase mutants having a neutral residue in position 134. The behavior of the E134D mutant shows that shortening the side chain of the nucleophile, despite maintaining a carboxylate group, confers glycosynthase activity. Therefore E134D is a transitional hydrolase to glycosynthase mutation.
- 出版日期2014-5-7
