Functional Mapping of Key Residues in Reductive Aminases Enabled by a High-Throughput RedAm Detect Assay

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Chemistry
• High-Throughput Screening

Background:

• Enzymatic reductive amination offers a sustainable route to chiral amines, crucial for pharmaceuticals and fine chemicals.
• Current enzyme engineering relies on slow, costly screening methods, hindering industrial application development.
• Optimizing single enzyme candidates is inefficient for broad applicability.

Purpose of the Study:

• To develop a versatile, high-throughput spectrophotometric/colorimetric assay for monitoring aminase activity.
• To enable rapid screening of enzyme libraries for improved reductive amination catalysts.
• To identify key residues influencing catalytic function and substrate specificity in reductive aminases (RedAms).

Main Methods:

• Development of the RedAm detect assay coupling product formation to an amine oxidase-HRP reporter system.
• Spectrophotometric monitoring of colored dye formation at 492/498 nm to quantify enzyme activity.
• Application of the assay to screen site saturation libraries of reductive aminases (RedAms), amine dehydrogenases (AmDHs), and amino acid dehydrogenases (AADHs).

Main Results:

• The RedAm detect assay successfully monitored reductive amination reactions catalyzed by RedAms, AmDHs, and AADHs.
• Screening of 56 site saturation libraries identified 10 key residues in BacRedAm critical for catalytic function and substrate specificity.
• Mutants with up to 7-fold improved activity were identified by targeting specific residues, demonstrating potential 'universal' hotspots for engineering substrate specificity.

Conclusions:

• The RedAm detect assay provides a rapid, reliable, and cost-effective method for high-throughput screening of aminase activity.
• This work establishes a foundation for mapping sequence-activity relationships in RedAms at the enzyme family level.
• The findings facilitate predictable, faster, and more cost-effective engineering of RedAms for diverse industrial applications.