Mechanism‐Guided Protein Engineering of <i>Paraburkholderia xenovorans</i> Lactamase for the Green Synthesis of 2‐(2‐Oxopyrrolidin‐1‐yl)‐Butanoic Acid

The synthesis of tertiary amides is crucial in pharmaceutical manufacturing, but achieving this efficiently through enzymatic methods remains challenging. Here, we describe the screening, evolution, and application of a lactamase for the synthesis of 2-(2-oxopyrrolidin-1-yl)-butanoic acid (2-OYBA) in aqueous phase. Through database mining, we identified a lactamase from Paraburkholderia xenovorans (PxAmpC) with the highest amide synthesis activity in this study at 31.3 U/g. Using protein crystallization, molecular dynamics simulations, and quantum mechanics calculations, we proposed and verified three potential synthesis mechanisms for 2-OYBA. We identified a mechanism for synthesis of 2-OYBA involving adenylation and lactamization, with the carboxyl group in the intermediate 2-2 (INT2-2) acting as a proton shuttle. Guided by this mechanism, rational design efforts enhanced amide synthesis activity by 16.6-fold. The PxAmpC mutants also exhibited a broader substrate scope for amide bond synthesis compared to the wild type. Our findings advance the green synthesis of complex amide compounds.