Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

Abstract

AbstractAbstract: A sustainable enzymatic synthesis of α‐keto acids from α‐amino acids deamination remains limited by the low catalytic efficiency of L‐amino acid deaminase (LAAD, EC 1.4.3.2). In this study, the 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 based on elucidation of catalytic mechanism. A protein engineering strategy was developed to shorten the distance D1 and get two variants W1 (exhibited for short‐chain aliphatic amino acids and charged amino acids) and W2( exhibited for large aromatic amino acids and sulfur‐containing amino acids). The reason is 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°. Finally, PmiLAAD variants (W1 and W2)were used in one pot in a parallel way, thus obtain strain S3, which exhibited conversion >90 % and titer >100 g/L toward six selected substrates. These results provide the basis for improving industrial production of α‐keto acids via microbial deamination of α‐amino acids.