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。

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-08-27 DOI:10.1007/s10930-023-10149-x
Mahdieh Mahboobi, Ali-Hatef Salmanian, Hamid Sedighian, Bijan Bambai
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引用次数: 1

摘要

简介:L-天冬酰胺酶(也称为L-ASNase)是一种重要的治疗酶,作为一种化疗药物,广泛用于治疗急性淋巴细胞白血病。此外,这种酶在食品工业中被用作食品加工试剂,以降低丙烯酰胺的含量。L-ASNase酶的活性和动力学参数的提高可能导致更高的效率,从而产生实际的成就。为了实现这一目标,我们选择88位的甘氨酸残基作为具有有利结果的潜在突变。方法:本研究首先对甘氨酸88进行了分子动力学模拟和分子对接等多种计算机分析,以寻找合适的突变位点。然后,采用合理的设计作为对酶进行分子修饰的最佳策略,以改善其酶性质。结果:我们的计算机研究结果表明,G88Q、G88L、G88K和G88A四个突变可能能够提高L-ASNase的天冬酰胺酶活性。每种酶的催化效率(kcat/Km)是比较突变体与野生型催化活性的最重要特征。实验室实验表明,G88Q突变体的kcat/Km比大肠杆菌K12 ASNase II(野生型)高36.32%,这表明低浓度的L-ASN可以提高L-ASNase的活性。突变体L-ASNase活性的动力学表征证实了与野生型酶相比,以ASN为底物的高周转率(kcat)。结论:计算机分析和实验室实验表明,G88Q突变而不是其他突变(G88L、G88K和G88A)可以改善L-ASNase的动力学。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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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.

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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
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.
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