Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Computational Biology

Background:

• Glycoside hydrolase family 13 (GH13) enzymes, including alpha-amylase and glucanotransferase, catalyze polysaccharide hydrolysis (e.g., glycogen, starch) and often exhibit transglycosylation activity.
• Understanding the structural basis of GH13 enzyme reaction specificity (transglycosylation vs. hydrolysis) is crucial for engineering enzymes for industrial, chemical, and biomedical applications.
• Current methods for identifying specificity determinants are limited, necessitating novel computational approaches.

Purpose of the Study:

• To develop and validate a computational method for decoding structural determinants that define GH13 enzyme reaction specificity.
• To identify residues, including those outside the active site, that influence the balance between transglycosylation and hydrolysis reactions.
• To engineer GH13 enzymes with altered reaction specificities using insights from the computational approach.

Main Methods:

• Proposed a computational approach based on the conservation of coevolving residues in spatial contacts linked to reaction specificity.
• Constructed enzyme variants of alpha-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima to test the algorithm's predictions.
• Utilized molecular dynamic simulations to analyze the structural and flexibility changes in enzyme variants.

Main Results:

• The K98P/D99A/H222Q variant of TmAmyA demonstrated a doubled transglycosylation/hydrolysis (T/H) ratio.
• The M279N variant of TmGTase exhibited a five-fold increase in the hydrolysis/transglycosidation ratio.
• Molecular dynamic simulations revealed that altered enzyme flexibility in variants correlates with the modified T/H ratios.

Conclusions:

• The developed computational approach effectively identifies key structural determinants of GH13 enzyme reaction specificity.
• The method successfully pinpointed residues outside the active site that significantly impact enzyme specificity.
• This strategy enables targeted engineering of GH13 enzymes for desired catalytic functions, with potential for biotechnological advancements.