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.
Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance
