Expression, purification and characterization of CTP synthase PyrG in Staphylococcus aureus

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-06-03 DOI:10.1016/j.pep.2024.106520

Dafeng Liu , Zhu Tian , Kuerban Tusong , Hayrinsa Mamat , Yihan Luo

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引用次数: 0

Abstract

Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.

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金黄色葡萄球菌中 CTP 合成酶 PyrG 的表达、纯化和表征

金黄色葡萄球菌（S. aureus）因其致病性，在医院和社区环境中都是一项重大挑战。耐药菌株的出现加剧了金黄色葡萄球菌感染，导致死亡率上升。PyrG 是三磷酸胞苷（CTP）合成酶家族的成员，由于 CTP 在细胞代谢中的关键作用，PyrG 成为了金黄色葡萄球菌的重要治疗靶点。然而，金黄色葡萄球菌 PyrG 的结构和机理细节仍然未知。在此，我们成功表达并纯化了单体 PyrG。根据分子对接的结果进行了突变实验。根据分子对接的结果，我们进行了突变实验，发现与野生型蛋白相比，Q386A显著降低了CTP合成酶的活性，而Y54A几乎完全取消了活性。将金黄色葡萄球菌暴露于激酶抑制剂克唑替尼会增加基因 pyrG 的表达。我们的研究结果确定了PyrG上CTP合成酶活性的两个关键位点，并发现PyrG基因表达在克唑替尼治疗期间有所增加，这可能最终为开发抗金葡菌感染的新药提供有价值的指导。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.