{"title":"通过计算机设计和体外定点突变优化II型大肠杆菌l-天冬氨酸酶活性的分子模型。","authors":"Mahdieh Mahboobi, Ali-Hatef Salmanian, Hamid Sedighian, Bijan Bambai","doi":"10.1007/s10930-023-10149-x","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><p>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.</p><h3>Method</h3><p>In this study, firstly to find the appropriate mutation on glycine 88, various <i>in silico</i> 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.</p><h3>Result</h3><p>Our <i>in silico</i> 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 (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the G88Q mutant is 36.32% higher than the <i>Escherichia coli</i> 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 (<i>k</i><sub>cat</sub>) with ASN as substrate relative to the wild type enzyme.</p><h3>Conclusion</h3><p><i>In silico analyses</i> and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2023-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis\",\"authors\":\"Mahdieh Mahboobi, Ali-Hatef Salmanian, Hamid Sedighian, Bijan Bambai\",\"doi\":\"10.1007/s10930-023-10149-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Introduction</h3><p>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.</p><h3>Method</h3><p>In this study, firstly to find the appropriate mutation on glycine 88, various <i>in silico</i> 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.</p><h3>Result</h3><p>Our <i>in silico</i> 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 (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the G88Q mutant is 36.32% higher than the <i>Escherichia coli</i> 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 (<i>k</i><sub>cat</sub>) with ASN as substrate relative to the wild type enzyme.</p><h3>Conclusion</h3><p><i>In silico analyses</i> and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.</p></div>\",\"PeriodicalId\":793,\"journal\":{\"name\":\"The Protein Journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2023-08-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"The Protein Journal\",\"FirstCategoryId\":\"2\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s10930-023-10149-x\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Protein Journal","FirstCategoryId":"2","ListUrlMain":"https://link.springer.com/article/10.1007/s10930-023-10149-x","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis
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 (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km 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 (kcat) 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.
期刊介绍:
The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.