Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of...

Area of Science:

• Carbohydrate Chemistry
• Enzymology
• Protein Engineering

Background:

• Glycobiology research is limited by the lack of synthetic, defined oligosaccharides.
• Enzyme-catalyzed glycosylation using glycoside hydrolases is hindered by their inherent hydrolytic activity.
• Current protein engineering methods for enzyme modification are often laborious and require extensive prior knowledge.

Purpose of the Study:

• To develop a straightforward and rapid strategy for engineering glycoside hydrolases to enhance transglycosylation yields.
• To identify single-mutant candidates that improve the efficiency of synthetic oligosaccharide production.
• To create a generic method applicable to various glycosidic bond formations and enzyme families.

Main Methods:

• Rational, rapid in silico analysis of protein sequences to predict beneficial mutations.
• Identification of 6-12 single-mutant candidates for improved transglycosylation.
• Validation of mutations in target enzymes and demonstration of their transposable nature.

Main Results:

• A novel in silico strategy successfully pinpointed single-mutant candidates to boost transglycosylation yields.
• The method requires minimal prior knowledge of the target enzyme, relying primarily on sequence data.
• Engineered enzymes demonstrated improved catalytic efficiency for forming diverse glycosidic bonds (pyranosides, furanosides; exo/endo action).
• Mutations were successfully transposed to other, even distantly related, enzymes, showcasing broad applicability.

Conclusions:

• This rational in silico approach offers a generic and efficient platform for engineering glycoside hydrolases.
• The method significantly accelerates the development of biocatalysts for synthesizing complex carbohydrates.
• The findings have broad implications for advancing glycobiology and carbohydrate-based therapeutics.