Enzymatic Mannich Reaction for the Synthesis of γ-Chiral Amino-Substituted α-Amino Acids

γ-Chiral amino-substituted α-amino acids are commonly found in bioactive molecules and play a critical role in drug design and development. However, the synthesis of these compounds remains challenging, as it requires protection of the reactive amino and carboxyl groups alongside stringent control over remote stereoselectivity. In this study, we developed a biocatalytic Mannich reaction catalyzed by engineered ApUstD, a characterized aldolase, enabling the synthesis of γ-chiral amino-substituted α-amino acids without the need for protection of the amino or carboxyl groups. Through protein engineering, the mutant ApUstD-M3 was obtained, showing good catalytic activity and selectivity, and also performing efficiently in preparative-scale reactions.