机制指导内酰胺酶的蛋白质工程改造用于绿色合成2-(氧代吡咯烷-1基)-丁酸

he amide bond plays a fundamental role in the synthesis of numerous pharmaceuticals. However, achieving efficient enzymatic synthesis of tertiary amides remains a persistent challenge. Here, we describe the screening, evolution and application of a lactamase aimed at synthesizing 2-(2-oxopyrrolidin-1-yl)-butanoic acid (2-OYBA) in aqueous phase. Initially, through database mining and evaluation, we identified a lactamase from Paraburkholderia xenovorans (PxAmpC) that exhibited the highest amide synthesis activity at 31.3 U/g. Further employing techniques such as protein crystallization, molecular dynamics (MD) simulations, and quantum mechanics (QM) calculations, we proposed and verified three potential synthesis mechanisms for synthesizing 2-OYBA. Subsequently, a rational target mechanism was found, involving adenylation where substrate carboxyl oxygen attacks α-P of ATP, forming intermediate 1 (INT1) with PPi and Mg2+ release; lactamization follows as amino group of INT1 attacks its ester, creating intermediate 2-2 (INT2-2), and the proton on INT2-2 amino group transfers to AMP phosphoric acid, forming production complex 1 (PC1). Notably, the carboxyl group in the substrate section of INT2-2 acts as a proton shuttle. Guided by this target mechanism, rational design efforts were implemented, resulting in a remarkable 16.6-fold enhancement in amide synthetic activity. The PxAmpC mutants demonstrated a broader substrate scope for amide bond synthesis compared to the wild type. Our findings provide insights into the green synthesis of complex amide compounds.