Sequence and Structure-Guided Engineering of Urethanase from Agrobacterium tumefaciens d3 for Improved Catalytic Activity

The amidase from Agrobacterium tumefaciens d3 (AmdA) degrades the carcinogenic ethyl carbamate (EC) in alcoholic beverages. However, its limited catalytic activity hinders practical applications. Here, multiple sequence alignment was first used to predict single variants with improved activity. Afterward, AlphaFold 2 was applied to predict the three-dimensional structure of AmdA and 21 amino acids near the catalytic triad were randomized by saturation mutagenesis. Each of the mutation libraries was then screened, and the improved single variants were combined to obtain the best double variant I97L/G195A that showed a 3.1-fold increase in the urethanase activity and a 1.5-fold increase in ethanol tolerance. MD simulations revealed that the mutations shortened the distance between catalytic residues and the substrate and enhanced the occurrence of a critical hydrogen bond in the catalytic pocket. This study displayed a useful strategy to engineer an amidase for the improvement of urethanase activity, and the variant obtained provided a good candidate for applications in the food industry.