N73-F124对在金黄色葡萄球菌MnSOD活性中的作用

Q4 Pharmacology, Toxicology and Pharmaceutics
D. Retnoningrum, H. Yoshida, M. D. Razani, V. F. Meidianto, A. Hartanto, A. Artarini, W. Ismaya
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引用次数: 3

摘要

细菌锰超氧化物歧化酶(MnSOD)是一种二聚体,负责其活性和稳定性。因此,增加二聚体强度将提高酶的稳定性,同时保持其活性。引入N73F取代以加强单体在二聚体界面上的相互作用。这种取代将在一个单体的F73与其他单体的F124之间引入π-堆积相互作用。用苯丙氨酸取代N73进行定点诱变。对突变体的活性进行定性和定量检查,同时用基于荧光的热位移测定法评估稳定性。最后,用X射线晶体学方法对突变体的结构进行了鉴定。N73F突变体的活性仅为野生型的约40%。N73F突变体在60+1°C时显示一个TM,而野生型有两个(在52-55°C和63-67°C时)。突变体的晶体结构显示了一种单体的F73与另一种单体的F124之间的相互作用。由于包括活性位点在内的酶结构没有变化,N73F结构呈现出一个谜。此外,N73和F124的位置和相互作用在人MnSOD中是保守的,但在氨基酸序列中有不同的位置。N73在酶活性中起作用,可能与其与F124的相互作用有关,F124位于活性位点区域,但尚未被认为参与反应。N73F取代揭示了N73-F124对在酶活性中前所未有的作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Unprecedented Role of the N73-F124 Pair in the Staphylococcus equorum MnSOD Activity
Bacterial manganese superoxide dismutase (MnSOD) occurs as a dimer, which is responsible for its activity and stability. Therefore, increasing the dimeric strength would increase the stability of the enzyme while maintaining its activity. An N73F substitution was introduced to strengthen interactions between the monomers at the dimer interface. This substitution would introduce a π-stacking interaction between F73 of one monomer to F124 from the other monomers. Site-directed mutagenesis was carried out to substitute N73 with phenylalanine. The activity of the mutant was qualitative- and quantitatively checked while the stability was evaluated with a fluorescence- based thermal-shift assay. Finally, the structure of the mutant was elucidated by means of Xray crystallography. The N73F mutant activity was only ~40% of the wild type. The N73F mutant showed one TM at 60+1°C while the wild type has two (at 52-55°C and 63-67°C). The crystal structure of the mutant showed the interactions between F73 from one monomer to F124 from the other monomer. The N73F structure presents an enigma because of no change in the enzyme structure including the active site. Furthermore, N73 and F124 position and interaction are conserved in human MnSOD but with a different location in the amino acid sequence. N73 has a role in the enzyme activity, likely related to its interaction with F124, which resides in the active site region but has not been considered to participate in the reaction. The N73F substitution has revealed the unprecedented role of the N73-F124 pair in the enzyme activity.
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来源期刊
Current Enzyme Inhibition
Current Enzyme Inhibition Pharmacology, Toxicology and Pharmaceutics-Drug Discovery
CiteScore
1.30
自引率
0.00%
发文量
30
期刊介绍: Current Enzyme Inhibition aims to publish all the latest and outstanding developments in enzyme inhibition studies with regards to the mechanisms of inhibitory processes of enzymes, recognition of active sites, and the discovery of agonists and antagonists, leading to the design and development of new drugs of significant therapeutic value. Each issue contains a series of timely, in-depth reviews written by leaders in the field, covering a range of enzymes that can be exploited for drug development. Current Enzyme Inhibition is an essential journal for every pharmaceutical and medicinal chemist who wishes to have up-to-date knowledge about each and every development in the study of enzyme inhibition.
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