Gene cloning, expression, and enzyme kinetics analysis of Eimeria tenella 2-methylcitrate synthase

文献类型： 外文期刊

第一作者： Gong, Zhenxing

作者： Gong, Zhenxing;Gong, Zhenxing;Qu, Zigang;Cai, Jianping;Gong, Zhenxing;Gong, Zhenxing;Gong, Zhenxing;Gong, Zhenxing;Cai, Jianping

作者机构：

关键词： Eimeria tenella; 2-methylcitrate synthase; Gene cloning and expression; Enzyme activity; Enzyme kinetic analysis

期刊名称：VETERINARY PARASITOLOGY （ 影响因子：2.6； 五年影响因子：2.5 ）

ISSN： 0304-4017

年卷期： 2024 年 328 卷

页码：

收录情况： SCI

摘要： In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH_ 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 mu g/reaction), with optimal enzyme activity observed at 41 degrees C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 +/- 0.17 mM, 1.102 +/- 0.08 mu M, and 5.999 +/- 1.24 mu M, respectively. The Vmax was 191.11 +/- 19.1 nmol/min/mg, 225.48 +/- 14.4 nmol/min/mg, and 370.02 +/- 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 mu g, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs.

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