Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China.
Parasitol Res. 2013 Apr;112(4):1719-27. doi: 10.1007/s00436-013-3330-6. Epub 2013 Feb 28.
Adenylate kinase 1 is responsible for the conversion of AMP into ADP involved in purine metabolism. In the present study, adenylate kinase 1 gene (CsADK1) was isolated from an adult cDNA library of Clonorchis sinensis, and the recombinant protein was expressed in Escherichia coli. Bioinformatics analysis implied that the putative protein contained 197 amino acids, and some residues in conservative binding sites of CsADK1 were substituted. The structure modeling analysis showed that CsADK1 was composed of a core domain, an NMP-binding domain, and a LID domain, which was just a small loop. It demonstrated that CsADK1 was a short isoform of ADKs. Moreover, CsADK1 was identified as an excretory/secretory product by western blot analysis. Real-time quantitative PCR showed that expression level of CsADK1 at the stage of excysted metacercaria was higher than those of adult worm (18.8-folds, P<0.01), metacercariae (1.5-folds, P<0.01), and eggs (5.6-folds, P<0.01). In addition, histochemistry analysis showed that CsADK1 was extensively distributed in metacercariae and in the vitellaria and eggs of adult worms. The Km and Vmax value for substrate ADP were 2.2 mM and 0.9 mM/min, respectively. The optimal temperature and pH value were 37 °C and from 7.5 to 8.0, respectively. The enzyme activity was highly dependent on Mg2+, and the optimal concentration of Mg2+ was 2 mM. However, the enzyme activity was slightly activated by Ca2+, and Mn2+ has no effect on activity. For monovalent ions, activity was highly activated by K+ and NH4+, but slightly by Li+. Taken together, CsADK1 was a metal ion-dependent enzyme involved in purine metabolism, which was important for development and reproduction, and might be a potential candidate for drug target for clonorchiasis.
腺嘌呤激酶 1 负责将 AMP 转化为 ADP，参与嘌呤代谢。本研究从华支睾吸虫成虫 cDNA 文库中分离出腺嘌呤激酶 1 基因（CsADK1），并在大肠杆菌中表达重组蛋白。生物信息学分析表明，该蛋白包含 197 个氨基酸，CsADK1 的一些保守结合位点的残基被取代。结构建模分析表明，CsADK1 由核心结构域、NMP 结合结构域和 LID 结构域组成，这只是一个小环。表明 CsADK1 是 ADK 的短同工酶。此外，Western blot 分析鉴定 CsADK1 为排泄/分泌产物。实时定量 PCR 显示，在囊蚴脱囊期 CsADK1 的表达水平高于成虫（18.8 倍，P<0.01）、囊蚴（1.5 倍，P<0.01）和虫卵（5.6 倍，P<0.01）。此外，组织化学分析显示，CsADK1 广泛分布于囊蚴和成虫的卵黄腺和卵中。CsADK1 对底物 ADP 的 Km 和 Vmax 值分别为 2.2 mM 和 0.9 mM/min。最适温度和 pH 值分别为 37°C 和 7.5-8.0。该酶的活性高度依赖于 Mg2+，最适 Mg2+浓度为 2 mM。然而，该酶的活性被 Ca2+轻微激活，Mn2+对活性没有影响。对于单价离子，活性被 K+和 NH4+高度激活，但被 Li+ 轻度激活。总之，CsADK1 是一种参与嘌呤代谢的金属离子依赖性酶，对发育和繁殖很重要，可能是华支睾吸虫病药物靶点的潜在候选物。
