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    A novel Amycolatopsis sulphurea L-alanine dehydrogenase with enhanced bidirectional catalysis: Rational design, expression, and biochemical characterization
    (Elsevier, 2026) Aksu, Tuba Okur; Sercinoglu, Onur; Aktas, Fatih; Binay, Baris
    The enzyme L-alanine Dehydrogenase (L-AlaDH) catalyzes the reversible conversion of pyruvate to L-alanine, utilizing NAD(H) and NAD+ as cofactors. Specifically, reductive amination facilitates the conversion of pyruvate to L-alanine in the presence of NAD(H). While numerous studies have reported on the oxidative activity of L-AlaDH, its reductive amination activity remains insufficiently explored. This study provides the first comprehensive characterization of the reductive amination activity of L-alanine dehydrogenase (L-AsAlaDH) from Amycolatopsis sulphurea. We found that engineered variants of the enzyme possess superior catalytic efficiency compared to the wild-type, a finding further underscored by their unexpected capacity for bidirectional conversion. Specifically, Tyr14Asn and Ile97Leu mutants, identified through rational enzyme engineering, were created, expressed, and purified. We determined the optimum pH, temperature, and kinetic parameters for both the deamination and amination activities of these enzymes. The effects of various metal ions on amination activity were also investigated. Statistical analysis was performed using two-way ANOVA and Dunnett's test. Furthermore, the storage stability of the reductive amination activity was assessed over a two-week period at-80,-20, +4, and + 25 degrees C. Ile97Leu exhibited approximately 3-fold higher catalytic efficiency (kcat/KM) in bidirectional reaction way compared to wild-type. We also identified the enhancing or inhibitory effects of various metal ions on the enzymes. Active site modelling elucidated that altered substrate and cofactor stability or flexibility contributed to the observed increases in catalytic efficiency. In conclusion, the enhanced reductive amination activity of the selected mutants suggests their potential as novel candidates for L-alanine production.