Reprogramming the Transition States to Enhance C–N Cleavage Efficiency of <i>Rhodococcus opacus</i> <scp>l</scp>-Amino Acid Oxidase
Yaoyun Wu, Yaozhong Cui, Wei Song, Wanqing Wei, Zhizhen He, Jinyang Tao, Dejing Yin, Xiulai Chen, Cong Gao, Jia Liu, Li Liu, Jing Wu
Abstract
High Resolution Image Download MS PowerPoint Slide 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.