Functional and structural insights into a thermostable (S)-selective amine transaminase and its improved substrate...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein Structure and Function
• Organic Chemistry

Background:

• Amine transaminases are crucial biocatalysts for synthesizing chiral amines.
• Improving enzyme stability and substrate scope is essential for industrial applications.
• Sbv333-ATA, a transaminase from Streptomyces, demonstrates high thermostability and broad substrate acceptance.

Purpose of the Study:

• To biochemically and structurally characterize Sbv333-ATA.
• To engineer Sbv333-ATA for an expanded substrate specificity, particularly for bulky amines.
• To elucidate the enzyme's active site and mechanism through structural analysis.

Main Methods:

• Biochemical assays to determine enzyme activity and stability.
• Crystallography to determine the 3D structures of the native enzyme and mutants.
• Site-specific mutagenesis to engineer enzyme properties.

Main Results:

• Sbv333-ATA exhibits a melting temperature of 85°C and stability in various organic solvents and biphasic systems.
• The enzyme accepts a range of amino donors, including amines and amino acids, but not sterically hindered aromatic amines.
• The W89A mutant showed significantly increased activity towards bulky diaromatic amines like 1,2-diphenylethylamine.
• High-resolution crystal structures revealed active site details and informed rational mutagenesis.

Conclusions:

• Sbv333-ATA is a robust biocatalyst with potential for diverse synthetic applications.
• Rational engineering, guided by structural insights, can effectively broaden the substrate specificity of transaminases.
• The W89A mutant represents a promising tool for the synthesis of amines previously inaccessible to Sbv333-ATA.