Biochemistry (Moscow)

, Volume 75, Issue 5, pp 655–664 | Cite as

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120

  • Ming Luo
  • Yong-Chao Guo
  • Jiao-Yu Deng
  • Hong-Ping Wei
  • Zhi-Ping Zhang
  • Yan Leng
  • Dong Men
  • Li-Rong Song
  • Xian-En Zhang
  • Ya-Feng ZhouEmail author


Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (Pi) during Pi starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during Pi starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase APA, coded by all2843, is characterized as a metalloenzyme containing Mg2+ and Zn2+ with molar ratio of 1: 2. Site-directed mutagenesis analysis indicated that, though the active center of APA is highly conserved in comparison with EAP, differences do exist between APA and EAP in metal ion coordination. Besides, biochemical analysis revealed that APA is a monomeric protein and inactivated rapidly at 50°C. These results suggest that APA is the first monomeric heat-labile classical PhoA found in cyanobacteria.

Key words

Anabaena sp. PCC7120 phosphorous starvation alkaline phosphatase metalloenzyme site-directed mutagenesis 



alkaline phosphatase


E. coli alkaline phosphatase


inductively coupled plasma-optical emission spectrometric analysis


para-nitrophenyl phosphate


alkaline phosphatase-encoding gene


product of phoA gene


quantitative polymerase chain reaction


streptavidin-binding peptide


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Wu, J., Sunda, W., Boyle, E. A., and Karl, D. M. (2000) Science, 289, 759–762.CrossRefPubMedGoogle Scholar
  2. 2.
    Sundareshwar, P. V., Morris, J. T., Koepfler, E. K., and Fornwalt, B. (2003) Science, 299, 563–565.CrossRefPubMedGoogle Scholar
  3. 3.
    Thingstad, T. F., Krom, M. D., Mantoura, R. F., Flaten, G. A., Groom, S., Herut, B., Kress, N., Law, C. S., Pasternak, A., Pitta, P., Psarra, S., Rassoulzadegan, F., Tanaka, T., Tselepides, A., Wassmann, P., Woodward, E. M., Riser, C. W., Zodiatis, G., and Zohary, T. (2005) Science, 309, 1068–1071.CrossRefPubMedGoogle Scholar
  4. 4.
    Vershinina, O. A., and Znamenskaia, L. V. (2002) Mikrobiologiya, 71, 581–595.Google Scholar
  5. 5.
    Coleman, J. E., and Gettins, P. (1983) Adv. Enzymol. Relat. Areas Mol. Biol., 55, 381–452.PubMedGoogle Scholar
  6. 6.
    Kim, E. E., and Wyckoff, H. W. (1991) J. Mol Biol., 218, 449–464.CrossRefPubMedGoogle Scholar
  7. 7.
    Stec, B., Holtz, K. M., and Kantrowitz, E. R. (2000) J. Mol. Biol., 299, 1303–1311.CrossRefPubMedGoogle Scholar
  8. 8.
    Orhanovic, S., and Pavela-Vrancic, M. (2003) Eur. J. Biochem., 270, 4356–4364.CrossRefPubMedGoogle Scholar
  9. 9.
    Derman, A. I., and Beckwith, J. (1991) J. Bacteriol., 173, 7719–7722.PubMedGoogle Scholar
  10. 10.
    Coleman, J. E., Nakamura, K., and Chlebowski, J. F. (1983) J. Biol. Chem., 258, 386–395.PubMedGoogle Scholar
  11. 11.
    Sowadski, J. M., Handschumacher, M. D., Murthy, H. M., Foster, B. A., and Wyckoff, H. W. (1985) J. Mol. Biol., 186, 417–433.CrossRefPubMedGoogle Scholar
  12. 12.
    Schopf, J. W. (1993) Science, 260, 640–646.CrossRefPubMedGoogle Scholar
  13. 13.
    Field, C. B., Behrenfeld, M. J., Randerson, J. T., and Falkowski, P. (1998) Science, 281, 237–240.CrossRefPubMedGoogle Scholar
  14. 14.
    Sanudo-Wilhelmy, S. A., Kustka, A. B., Gobler, C. J., Hutchins, D. A., Yang, M., Lwiza, K., Burns, J., Capone, D. G., Raven, J. A., and Carpenter, E. J. (2001) Nature, 411, 66–69.CrossRefPubMedGoogle Scholar
  15. 15.
    Watson, G. M., Scanlan, D. J., and Mann, N. H. (1996) FEMS Microbiol. Lett., 142, 105–109.CrossRefPubMedGoogle Scholar
  16. 16.
    Hirani, T. A., Suzuki, I., Murata, N., Hayashi, H., and Eaton-Rye, J. J. (2001) Plant Mol. Biol., 45, 133–144.CrossRefPubMedGoogle Scholar
  17. 17.
    Suzuki, S., Ferjani, A., Suzuki, I., and Murata, N. (2004) J. Biol. Chem., 279, 13234–13240.CrossRefPubMedGoogle Scholar
  18. 18.
    Block, M. A., and Grossman, A. R. (1988) Plant Physiol., 86, 1179–1184.CrossRefPubMedGoogle Scholar
  19. 19.
    Bone, D. H. (1971) Arch. Microbiol., 80, 147–153.Google Scholar
  20. 20.
    Ray, J. M., Bhaya, D., Block, M. A., and Grossman, A. R. (1991) J. Bacteriol., 173, 4297–4309.PubMedGoogle Scholar
  21. 21.
    Doonan, B. B., and Jensen, T. E. (1980) Microbios, 29, 185–207.PubMedGoogle Scholar
  22. 22.
    Wagner, K. U., Masepohl, B., and Pistorius, E. K. (1995) Microbiology, 141(Pt. 12), 3049–3058.CrossRefPubMedGoogle Scholar
  23. 23.
    Su, Z. C., Olman, V., and Xu, Y. (2007) Bmc Genomics, 8, 156–167.CrossRefPubMedGoogle Scholar
  24. 24.
    Ihlenfeldt, M. J. A., and Gibson, J. (1975) Arch. Microbiol., 102, 23–28.CrossRefPubMedGoogle Scholar
  25. 25.
    Grainger, S. L., Peat, A., Tiwari, D. N., and Whitton, B. A. (1989) Microbios, 59, 7–17.PubMedGoogle Scholar
  26. 26.
    Kumar, A., Singh, S., and Tiwari, D. N. (1992) World J. Microbiol. Biotechnol., 8, 585–588.CrossRefGoogle Scholar
  27. 27.
    Kaneko, T., Nakamura, Y., Wolk, C. P., Kuritz, T., Sasamoto, S., Watanabe, A., Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takazawa, M., Yamada, M., Yasuda, M., and Tabata, S. (2001) DNA Res., 8, 205–213, 227–253.CrossRefPubMedGoogle Scholar
  28. 28.
    Ning, D., and Xu, X. (2004) Microbiology, 150, 447–453.CrossRefPubMedGoogle Scholar
  29. 29.
    Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics, 23, 2947–2948.CrossRefPubMedGoogle Scholar
  30. 30.
    Guindon, S., and Gascuel, O. (2003) Syst. Biol., 52, 696–704.CrossRefPubMedGoogle Scholar
  31. 31.
    Felsenstein, J. (1985) Evolution, 39, 783–791.CrossRefGoogle Scholar
  32. 32.
    Sebastian, M., and Ammerman, J. W. (2009) ISME J., 3, 563–572.CrossRefPubMedGoogle Scholar
  33. 33.
    Gierasch, L. M. (1989) Biochemistry, 28, 923–930.CrossRefPubMedGoogle Scholar
  34. 34.
    Wojciechowski, C. L., and Kantrowitz, E. R. (2002) J. Biol. Chem., 277, 50476–50481.CrossRefPubMedGoogle Scholar
  35. 35.
    Stec, B., Hehir, M. J., Brennan, C., Nolte, M., and Kantrowitz, E. R. (1998) J. Mol. Biol., 277, 647–662.CrossRefPubMedGoogle Scholar
  36. 36.
    Chaidaroglou, A., Brezinski, D. J., Middleton, S. A., and Kantrowitz, E. R. (1988) Biochemistry, 27, 8338–8343.CrossRefPubMedGoogle Scholar
  37. 37.
    Xu, X., and Kantrowitz, E. R. (1993) Biochemistry, 32, 10683–10691.CrossRefPubMedGoogle Scholar
  38. 38.
    Xu, X., and Kantrowitz, E. R. (1992) J. Biol. Chem., 267, 16244–16251.PubMedGoogle Scholar
  39. 39.
    Tibbitts, T. T., Xu, X., and Kantrowitz, E. R. (1994) Protein Sci., 3, 2005–2014.CrossRefPubMedGoogle Scholar
  40. 40.
    Martin, D. C., Pastra-Landis, S. C., and Kantrowitz, E. R. (1999) Protein Sci., 8, 1152–1159.CrossRefPubMedGoogle Scholar
  41. 41.
    Ma, L., and Kantrowitz, E. R. (1994) J. Biol. Chem., 269, 31614–31619.PubMedGoogle Scholar
  42. 42.
    Kozlenkov, A., Manes, T., Hoylaerts, M. F., and Millan, J. L. (2002) J. Biol. Chem., 277, 22992–22999.CrossRefPubMedGoogle Scholar
  43. 43.
    Tibbitts, T. T., Murphy, J. E., and Kantrowitz, E. R. (1996) J. Mol. Biol., 257, 700–715.CrossRefPubMedGoogle Scholar
  44. 44.
    Chaidaroglou, A., and Kantrowitz, E. R. (1989) Protein Eng., 3, 127–132.CrossRefPubMedGoogle Scholar
  45. 45.
    Matlin, A. R., Kendall, D. A., Carano, K. S., Banzon, J. A., Klecka, S. B., and Solomon, N. M. (1992) Biochemistry, 31, 8196–8200.CrossRefPubMedGoogle Scholar
  46. 46.
    Xu, X., and Kantrowitz, E. R. (1991) Biochemistry, 30, 7789–7796.CrossRefPubMedGoogle Scholar
  47. 47.
    Xu, H., Zhang, X., Zhang, Z., Zhang, Y., and Cass, A. E. G. (2003) Biocatal. Biotransform., 21, 41–47.CrossRefGoogle Scholar
  48. 48.
    Bradshaw, R. A., Cancedda, F., Ericsson, L. H., Neumann, P. A., Piccoli, S. P., Schlesinger, M. J., Shriefer, K., and Walsh, K. A. (1981) Proc. Natl. Acad. Sci. USA, 78, 3473–3477.CrossRefPubMedGoogle Scholar
  49. 49.
    Le Du, M. H., Stigbrand, T., Taussig, M. J., Menez, A., and Stura, E. A. (2001) J. Biol. Chem., 276, 9158–9165.CrossRefPubMedGoogle Scholar
  50. 50.
    Helland, R., Larsen, R. L., and Asgeirsson, B. (2009) Biochim. Biophys. Acta, 1794, 297–308.PubMedGoogle Scholar
  51. 51.
    Janeway, C. M., Xu, X., Murphy, J. E., Chaidaroglou, A., and Kantrowitz, E. R. (1993) Biochemistry, 32, 1601–1609.CrossRefPubMedGoogle Scholar
  52. 52.
    Zappa, S., Rolland, J. L., Flament, D., Gueguen, Y., Boudrant, J., and Dietrich, J. (2001) Appl. Environ. Microbiol., 67, 4504–4511.CrossRefPubMedGoogle Scholar
  53. 53.
    Boulanger, R. R., Jr., and Kantrowitz, E. R. (2003) J. Biol. Chem., 278, 23497–23501.CrossRefPubMedGoogle Scholar
  54. 54.
    Nakashima, H., Fukuchi, S., and Nishikawa, K. (2003) J. Biochem., 133, 507–513.CrossRefPubMedGoogle Scholar
  55. 55.
    Thornton, J. M. (1981) J. Mol. Biol., 151, 261–287.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • Ming Luo
    • 1
    • 2
  • Yong-Chao Guo
    • 1
  • Jiao-Yu Deng
    • 1
  • Hong-Ping Wei
    • 1
  • Zhi-Ping Zhang
    • 1
  • Yan Leng
    • 1
    • 2
  • Dong Men
    • 1
    • 2
  • Li-Rong Song
    • 3
  • Xian-En Zhang
    • 1
  • Ya-Feng Zhou
    • 1
    Email author
  1. 1.State Key Laboratory of Virology, Wuhan Institute of VirologyChinese Academy of SciencesWuhanChina
  2. 2.Graduate SchoolChinese Academy of ScienceBeijingChina
  3. 3.State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of HydrobiologyChinese Academy of SciencesWuhanChina

Personalised recommendations