Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Biotechnology

Background:

• NAD(P)+-dependent L-amino acid dehydrogenases are widely used in synthesis and sensing.
• The biotechnological application of D-amino acid dehydrogenase (D-AADH) has been limited due to the lack of suitable enzymes from natural sources.

Purpose of the Study:

• To engineer a functional D-amino acid dehydrogenase (D-AADH) using protein engineering methods.
• To explore the application of the engineered D-AADH in the synthesis and analysis of D-amino acids.

Main Methods:

• Protein engineering of meso-diaminopimelate dehydrogenase (meso-DAPDH) to create D-AADH.
• Characterization of the engineered D-AADH's catalytic activity and stability.
• Application of D-AADH in the synthesis of D-branched-chain amino acids (D-BCAAs) and isotopic labeling.
• Development of an assay for D-isoleucine detection.

Main Results:

• An NADP+-dependent D-AADH was successfully created from meso-DAPDH.
• The engineered D-AADH efficiently catalyzed the oxidative deamination of D-amino acids to 2-oxo acids.
• The enzyme demonstrated high yields and optical purity in the synthesis of D-BCAAs, including 13C- and/or 15N-labeled variants.
• The D-AADH enabled selective assay of D-isoleucine in complex mixtures.

Conclusions:

• Protein engineering provides an effective strategy for creating novel D-AADH with desired biotechnological applications.
• The engineered D-AADH is a valuable tool for the synthesis of D-amino acids and the development of diagnostic methods.
• Structural analysis and mutation design can significantly enhance enzyme activity and tailor reactivity profiles.