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Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

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Abstract

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40°C, respectively. The calculated activation energy is 56.04 kJ mol−1. The Km of the CsAK3 for AMP and GTP are 118 μM and 359 μM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.

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References

  • Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brochiero E, Coady MJ, Klein H, Laprade R, Lapointe JY (2001) Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules. Biochim Biophys Acta 1510:29–42

    Google Scholar 

  • Cabantchik ZI (1990) Properties of permeation pathways induced in the human red cell membrane by malaria parasites. Blood Cells 16:421–32

    Google Scholar 

  • Chung CS, Lee SK (1976) An epidemiological study of primary liver carcinomas in Busan area with special reference to clonorchiasis. Korean J Pathol 10:33–46

    Google Scholar 

  • Criswell AR, Bae E, Stec B, Konisky J, Phillips GN (1994) Structures of thermophilic and mesophilic adenylate kinases from the genus Methanococcus. J Mol Biol 330:1087–1099

    Google Scholar 

  • Crompton DW (1999) How much human helminthiasis is there in the world? J Parasitol 85:397–403

    CAS  PubMed  Google Scholar 

  • Dahlem YA, Horn TFW, Buntinas L, Gonoi T, Wolf G, Siemen D (2004) The human mitochondrial KATP channel is modulated by calcium and nitric oxide: a patch-clamp approach. Biochim Biophys Acta 1656:46–56

    Google Scholar 

  • Dzeja PP, Bortolon R, Perez-Terzic C, Holmuhamedov EL, Terzic A (2002) Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer. Proc Natl Acad Sci U S A 99:10156–10161

    Google Scholar 

  • Fukami-Kobayashi K, Nosaka M, Nakazawa A, Go M (1996) Ancient divergence of long and short isoforms of adenylate kinase: molecular evolution of the nucleoside monophosphate kinase family. FEBS Lett 385:214–220

    Google Scholar 

  • Heldt HW, Schwalbach K (1967) The participation of GTP-AMP-P transferase in substrate level phosphate transfer of rat liver mitochondria. Eur J Biochem 1:199–206

    Google Scholar 

  • Illanes A, Wilson L, Tomasello G (2001) Effect of modulation of enzyme inactivation on temperature optimization for reactor operation with chitin-immobilized lactase. J Mol Catal B Enzym 11:531–540

    Google Scholar 

  • Inouye S, Yamada Y, Miura K, Suzuki H, Kawata K, Shinoda K, Nakazawa A (1999) Distribution and developmental changes of adenylate kinase isozymes in the rat brain: localization of adenylate kinase 1 in the olfactory bulb. Biochem Biophys Res Commun 254:618–622

    Google Scholar 

  • Janssen E, Terzic A, Wieringa B, Dzeja PP (2003) Impaired intracellular energetic communication in muscles from creatine kinase and adenylate kinase (M-CK/AK1) double knockout mice. J Biol Chem 278:30441–30449

    Google Scholar 

  • Kohler C, Gahm A, Noma T, Nakazawa A, Orrenius S, Zhivotovsky B (1999) Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis. FEBS Lett 447:10–12

    Google Scholar 

  • Konrad M (1993) Identification and characterization of a yeast gene encoding an adenylate kinase homolog. Biochim Biophys Acta 1172:12–16

    Google Scholar 

  • Kuby SA, Hamada M, Gerber D, Tsai WC, Jacobs HK, Cress MC, Chua GK, Fleming G, Wu LH, Fischer AH, Frischat A, Maland L (1978) Studies on adenosine triphosphate transphosphorylases, isolation and several properties of the crystalline calf ATP-AMP transphosphorylases (adenylate kinases) from muscle and liver and some observations on the rabbit muscle adenylate kinase. Arch Biochem Biophys 187:34–52

    Google Scholar 

  • Lee JH, Yang HM, Bak UB, Rim HJ (1994) Promoting role of Clonorchis sinensis infection on induction of cholangiocarcinoma during two-step carcinogenesis. Korean J Parasitol 32:13–18

    CAS  PubMed  Google Scholar 

  • Li X, Pan XM (2000) Both native conformers of rabbit muscle adenylate kinase are active. FEBS Lett 480:235–238

    Google Scholar 

  • Miura K, Inouye S, Sakai K, Takaoka H, Kishi F, Tabuchi M, Tanaka T, Matsumoto H, Shirai M, Nakazawa T, Nakazawa A (2001) Cloning and characterization of adenylate kinase from Chlamydia pneumoniae. J Biol Chem 276:13490–13498

    Google Scholar 

  • Miyazaki K, Wintrode PL, Grayling RA, Rubingh DN, Arnold FH (2000) Directed evolution study of temperature adaptation in a psychrophilic enzyme. J Mol Biol 297:1015-1026

    Google Scholar 

  • Muller CW, Schlauderer GJ, Reinstein J, Schulz GE (1996) Adenylate kinase motions during catalysis: an energetic counterweight balancing substrate binding. Structure 4:147–156

    Google Scholar 

  • Noma T, Fujisawa K, Yamashiro Y, Shinohara M, Nakazawa A, Gondo T, Ishihara T, Yoshinobu K (2001) Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix. Biochem J 358:225–232

    Google Scholar 

  • Pai EF, Schulz GE, Tomasselli AG, Noda LH (1983) Preliminary X-ray studies on the GTP: AMP phosphotransferase from beef heart mitochondria. J Mol Biol 164:347–350

    Google Scholar 

  • Schricker R, Magdolen V, Bandlow W (1992) A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria. Mol Gen Genet 233:363–371

    Google Scholar 

  • Schricker R, Magdolen V, Strobel G, Bogengruber E, Breitenbach M, Bandlow W (1995) Strain-dependent occurrence of functional GTP: AMP phosphotransferase (AK3) in saccharomyces cerevisiae. J Biol Chem 270:31103–31110

    Google Scholar 

  • Schricker R, Angermayr M, Strobel G, Klinke S, Korber D, Bandlow W (2002) Redundant mitochondrial targeting signals in yeast adenylate kinase. J Biol Chem 277:28757–28764

    Google Scholar 

  • Schulz GE, Schiltz E, Tomasselli AG, Frank R, Brune M, Wittinghofer A, Schirmer RH (1986) Structural relationships in the adenylate kinase family. Eur J Biochem. 161:127–132

    Google Scholar 

  • Sheng X, Pan X, Wang C, Zhang Y, Jing G (2001) Conformational and functional significance of residue proline 17 in chicken muscle adenylate kinase. FEBS Lett 508:318-322

    Google Scholar 

  • Tomasselli AG, Schirmer RH, Noda LH (1979) Mitochondrial GTP-AMP phosphotransferase 1, purification and properties. Eur J Biochem 93:257–262

    Google Scholar 

  • Tugarinov V, Shapiro YE, Liang Z, Freed JH, Meirovitch E (2002) A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling (15) N NMR relaxation. J Mol Biol 315:155–170

    Google Scholar 

  • Ulschmid JK, Rahlfs S, Schirmer RH, Becker K (2004) Adenylate kinase and GTP: AMP phosphotransferase of the malarial parasite Plasmodium falciparum central players in cellular energy metabolism. Mol Biochem Parasitol 136:211–220

    Google Scholar 

  • Van Rompay AR, Johansson M, Karlsson A (2000) Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases. Pharmacol Ther 87:189–198

    Google Scholar 

  • Vonrhein C, Schlauderer GJ, Schulz GE (1995) Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases. Structure 3:483–490

    Google Scholar 

  • Yamada M, Sugahara M, Hishitani Y, Nobumoto M, Nakazawa A (1998) Isolation and characterization of mutated mitochondrial GTP: AMP phosphotransferase. J Mol Biol 280:551–558

    Google Scholar 

Download references

Acknowledgements

This research was funded by grants from the Natural Science Foundation of Guangdong Province (team program), China; the office of Health and the office of Science and Technology of Guangdong Province, China (no. 2002B31005); the key Science and Technique program of Guangzhou city, Guangdong Province, China (no. 200223-E4022). The experiments comply with the current laws of the country in which the experiments were performed.

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Authors and Affiliations

  1. Department of Parasitology, Medical School, Sun Yat-Sen University, 74 Zhongshan 2 Road, 510089, Guangzhou , P.R. China

    Guang Yang, Xinbing Yu, Zhongdao Wu, Jin Xu, Linxia Song, Hongmei Zhang, Xuchu Hu, Nancai Zheng & Jian Xu

  2. Key Laboratory for Tropical Diseases Control, Ministry of Education, 510089, Guangzhou , P.R. China

    Xinbing Yu, Zhongdao Wu, Jin Xu, Xuchu Hu & Jian Xu

  3. State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 200433, Shanghai , P.R. China

    Guang Yang, Jin Xu, Lingchen Guo, Jian Xu, Jianfeng Dai, Chaoneng Ji, Shaohua Gu & Kang Ying

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  1. Guang Yang
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  2. Xinbing Yu
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  3. Zhongdao Wu
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Correspondence to Xinbing Yu.

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Yang, G., Yu, X., Wu, Z. et al. Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis. Parasitol Res 95, 406–412 (2005). https://doi.org/10.1007/s00436-005-1305-y

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  • DOI: https://doi.org/10.1007/s00436-005-1305-y

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