Reprogramming the Transition States to Enhance C-N Cleavage Efficiency of Rhodococcus opacus l-Amino Acid Oxidase.

l - Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus ( Ro LAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [ TS1 ] in step 1 and Int1 → [ TS2 ] in step 2. We reprogrammed the transition states [ TS1 ] and [ TS2 ] to reduce the identified energy barrier and obtain a Ro LAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12-24 h, in a 5 L reactor. Our results indicated the promising potential of the developed Ro LAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.