Development of an Enzyme Cascade System for the Synthesis of Enantiomerically Pure D-Amino Acids Utilizing Ancestral L-Amino Acid Oxidase.

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH4Cl. Concurrently, any generated H2O2 is decomposed into O2 and H2O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.

Definition of an Index Parameter To Screen Highly Functional Enzymes Derived from a Biochemical and Thermodynamic Analysis of Ancestral meso-Diaminopimelate Dehydrogenases.

Sequence-based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time-consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso-2,6-diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH-N1, -N2, -N3, and -N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH-N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that "quality of mutations" is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH-N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high-functioning enzymes from sequence data.