Structure‐Guided Engineering of a Ser192Ala Variant Enhances Bidirectional Catalysis in Amycolatopsis sulphurea L‐Alanine Dehydrogenase

ABSTRACT

L‐alanine dehydrogenase (L‐AlaDH) catalyzes the reversible conversion of pyruvate to L‐alanine, mediated by the NAD ⁺ /NADH cofactor pair. In this reaction, reductive amination converts pyruvate into L‐alanine in the presence of NADH. While oxidative deamination is widely documented, the reductive amination activity of L‐AlaDH remains less thoroughly characterized. In this study, we performed a comprehensive characterization of L‐AlaDH from Amycolatopsis sulphurea ( As AlaDH) and an engineered variant, Ser192Ala, developed through rational design. The variant was expressed, purified, and evaluated across both reaction directions. We established optimal pH and temperature profiles and determined kinetic parameters, revealing that the Ser192Ala substitution yielded an approximately two‐fold increase in catalytic efficiency ( k _cat /K _M ) compared to the wild‐type enzyme for both amination and deamination. We investigated the influence of metal ions (Zn ^{2 +} , Fe ^{3 +} , K ⁺ , Na ⁺ , Li ⁺ , Mg ^{2 +} , Ca ^{2 +} ) on amination activity using two‐way ANOVA and Dunnett's post‐hoc analysis, and assessed storage stability over two weeks at temperatures ranging from –80°C to 20°C. Structural analysis and molecular dynamics simulations suggested improved substrate and cofactor positioning, along with altered active site flexibility, likely contributed to the enhanced catalytic efficiency. Overall, the superior reductive amination activity of the Ser192Ala variant highlights its potential as a robust biocatalyst for industrial L‐alanine production.