Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis

• Published in: The Protein Journal Vol. 42; no. 6; pp. 664 - 674
• Main Authors: Mahboobi, Mahdieh, Salmanian, Ali-Hatef, Sedighian, Hamid, Bambai, Bijan
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.12.2023; Springer Nature B.V
• Subjects: Acrylamide; Acute lymphoblastic leukemia; Animal Anatomy; Asparaginase; Biochemistry; Bioorganic Chemistry; Catalytic activity; Chemical reduction; Chemistry; Chemistry and Materials Science; E coli; Enzymes; Food industry; Food processing; Food processing industry; Glycine; Histology; L-asparaginase; Leukemia; Lymphatic leukemia; Molecular docking; Molecular modelling; Morphology; Mutants; Mutation; Optimization; Organic Chemistry; Reagents; Site-directed mutagenesis; Substrates; Turnover rate; L-asparaginase II; In silico; Kinetic characterization; Catalytic activity; Site-directed mutagenesis
• ISSN: 1572-3887; 1875-8355; 1573-4943
• DOI: 10.1007/s10930-023-10149-x

Introduction L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. Method In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. Result Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase’s asparaginase activity. The catalytic efficiency of each enzyme ( k cat / K m ) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the k cat / K m for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate ( k cat ) with ASN as substrate relative to the wild type enzyme. Conclusion In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.