Crystal structure of urethanase from Candida parapsilosis and insights into the substrate-binding through in silico mutagenesis and improves the catalytic activity and stability.

Ethyl carbamate (EC) is classified as a Class 2A carcinogen, and is present in various fermented foods, posing a threat to human health. Urethanase (EC 3.5.1.75) can catalyze EC to produce ethanol, CO2 and NH3. The urethanase (cpUH) from Candida parapsilosis can hydrolyze EC, but its low affinity and poor stability hinder its application. Here, the structure of cpUH from Candida parapsilosis was determined with a resolution of 2.66 Å. Through sequence alignment and site-directed mutagenesis, it was confirmed that cpUH contained the catalytic triad Ser-cisSer-Lys of the amidase family. Then, the structure-oriented engineering mutant N194V of urethanase was obtained. Its urethanase activity increased by 6.12 %, the catalytic efficiency (kcat/Km) increased by 21.04 %, and the enzyme stability was also enhanced. Modeling and molecular docking analysis showed that the variant N194V changed the number of hydrogen bonds between the substrate and the catalytic residue, resulting in enhanced catalytic ability. MD simulation also demonstrated that the introduction of hydrophobic amino acid Val reduced the RMSD value and increased protein stability. The findings of this study suggest that the N194V variant exhibits significant potential for industrial applications due to its enhanced affinity for substrate binding, improved catalytic efficiency, and increased enzyme stability.