Novel Thermus thermophilus L-Alanine dehydrogenase mutants: Synthesis of L-alanine derivatives with reductive amination

Abstract

Non-proteinogenic amino acids are valuable compounds for pharmaceutical and chemical applications. When enzymatic synthesis offers a sustainable and enantioselective alternative to chemical methods, the reductive amination potential of L-alanine dehydrogenases has been investigated for activity on larger keto acids. This study presents the first report on the engineering of Thermus thermophilus L-alanine dehydrogenase (TtAlaDH) to enhance its reductive amination activity for α-ketovalerate and α-ketocaproate, broadening its substrate scope beyond its natural pyruvate preference. Using active-site redesigning technique, the Tyr92 residue of TtAlaDH was targeted, and Tyr92Ser mutant with significantly improved activity was generated. Kinetic analysis demonstrated 198-fold increase in kcat and 30-fold rise in KM for α-ketocaproate, resulting in 6.6-fold enhancement in catalytic efficiency (kcat/KM). Similarly, for α-ketovalerate, kcat/KM increased 1.7-fold. The activity for smaller substrates such as α-ketobutyrate and pyruvate declined. Molecular modeling revealed that the Tyr92Ser mutation remodeled the active site enabling enhanced reductive amination. This is the first study demonstrating the successful synthesis of L-2-aminobutyrate, L-norvaline, and L-norleucine via enzymatic reductive amination using an engineered L-AlaDH, achieving conversion 43 %, 47 %, and 70 % yields, respectively, with >99 % enantiopurity. This work establishes a novel biocatalytic approach for the green synthesis of valuable L-alanine derivatives, highlighting its industrial potential.