Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Structural Biology

Background:

• D-amino acid oxidase (DAAO) is an enzyme that degrades D-amino acids, producing α-ketoacids, hydrogen peroxide, and ammonia.
• DAAO has significant potential for industrial and clinical applications, driving interest in its engineering.

Purpose of the Study:

• To engineer mammalian DAAOs (pig and human) by combining literature data with detailed structural analysis.
• To investigate the role of specific non-obvious residues in modulating DAAO substrate specificity, activity, and product release.

Main Methods:

• Detailed structural analysis of DAAO.
• Molecular dynamics simulations to study solvent accessibility and product release.
• Site-directed mutagenesis of critical residues identified in the active site and secondary binding pocket.
• Biochemical characterization of engineered DAAO variants.

Main Results:

• Identified critical residues on loops bordering the active site and secondary binding pocket that influence substrate specificity and activity in pig and human DAAO.
• Engineered DAAO variants with altered biochemical properties through targeted mutations.
• Demonstrated the importance of these residues in controlling substrate specificity, product egress, and overall enzyme activity.

Conclusions:

• The identified residues are crucial for modulating DAAO's function, including substrate specificity and product release.
• This study provides a foundation for further re-engineering of DAAO for optimized clinical and industrial applications.