L-amino acid deaminase (LAAD, EC 1.4.3.2) catalyzes the deamination of α-amino acids. At present, sustainable enzymatic α-keto acids synthesis remains limited by the low catalytic efficiency of wild-type LAADs. In this study, catalytic mechanism was elucidated, and catalytic distance D1 between the substrate αC-H and the cofactor FAD N(5) was identified as the key factor limiting efficiency of Proteus mirabilis PmiLAAD. Shortening the distance via protein engineering improved catalytic efficiency toward six selected amino acids. The two variants with the best catalytic properties were W1, which exhibited a preference for short-chain aliphatic amino acids and charged amino acids, and W2, which showed a preference for large aromatic amino acids and sulfur-containing amino acids. The mutated residues in the two variants altered the binding pose of the substrate, α-hydrogen was improved to be more perpendicular against the plain of the isoalloxazine ring causing the angle between the substrates’ αC-H, FAD N(5), and FAD N(10) to approach 90°, and thus shortened the distance. Finally, W1 and W2 were cascade in one Escherichia coli cell to obtain strain S3, which exhibited conversion >90% and yield >100 g/L toward all selected substrates. These results provide the basis for improving industrial production of α-keto acids via microbial deamination of α-amino acids.
