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Crystal structure of urethanase from Candida parapsilosis and insights into the substrate-binding through in silico mutagenesis and improves the catalytic activity and stability.
Yang, Lijuan; Zhao, Ting; Zhang, Xian; Fan, Tingting; Zhang, Yao; Feng, Zhiping; Liu, Jun.
Afiliación
  • Yang L; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China; Liquor Making Bio-Technology & Application of Key Laboratory of Sichuan Province, Sichuan University of Science & Engineering, Yibin, China. Electronic address: yanglijuan@suse.edn.cn.
  • Zhao T; School of Pharmaceutical Sciences, Wuhan University, Wuhan, China. Electronic address: zhaoting@wuh.edu.cn.
  • Zhang X; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China.
  • Fan T; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China.
  • Zhang Y; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China.
  • Feng Z; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China; Liquor Making Bio-Technology & Application of Key Laboratory of Sichuan Province, Sichuan University of Science & Engineering, Yibin, China. Electronic address: fengzhiping@suse.edn.cn.
  • Liu J; College of Bioengineering, Sichuan University of Science & Engineering, Yinbin, China; Liquor Making Bio-Technology & Application of Key Laboratory of Sichuan Province, Sichuan University of Science & Engineering, Yibin, China. Electronic address: liujunbio@suse.edu.cn.
Int J Biol Macromol ; 278(Pt 2): 134763, 2024 Oct.
Article en En | MEDLINE | ID: mdl-39151849
ABSTRACT
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Estabilidad de Enzimas / Simulación del Acoplamiento Molecular / Candida parapsilosis Idioma: En Revista: Int J Biol Macromol Año: 2024 Tipo del documento: Article Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Estabilidad de Enzimas / Simulación del Acoplamiento Molecular / Candida parapsilosis Idioma: En Revista: Int J Biol Macromol Año: 2024 Tipo del documento: Article Pais de publicación: Países Bajos