title: Identification and biochemical characterization of adenylate kinase 1 from Clonorchis sinensis.
abstract: 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.
authors: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, China.
authors: Pei
authors: P
authors: Liang
authors: Fan
authors: F
authors: Zhang
authors: Wenjun
authors: W
authors: Chen
authors: Xuchu
authors: X
authors: Hu
authors: Yan
authors: Y
authors: Huang
authors: Shan
authors: S
authors: Li
authors: Mengyu
authors: M
authors: Ren
authors: Lei
authors: L
authors: He
authors: Ran
authors: R
authors: Li
authors: Xuerong
authors: X
authors: Li
authors: Jin
authors: J
authors: Xu
authors: Zhongdao
authors: Z
authors: Wu
authors: Gang
authors: G
authors: Lu
authors: Xinbing
authors: X
authors: Yu
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics*; D019295:Computational Biology; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D008670:Metals; D000644:Quaternary Ammonium Compounds; D000244:Adenosine Diphosphate; D000263:Adenylate Kinase; C110939:adenylate kinase 1