Science in China Series C: Life Sciences

, Volume 51, Issue 12, pp 1066–1075 | Cite as

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

  • Jie Zhou
  • JunXia Zhou
  • HaoMeng Yang
  • ChengShi Yan
  • Fang Huang
Article

Abstract

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na+. Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.

Keywords

cyanobacteria Synechocystis putative glutaminase enzyme activity mutagenesis salt tolerance 

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References

  1. 1.
    Nandakumar R, Yoshimune K, Wakayama M, et al. Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry. J Mol Catal B Enzymes, 2003, 23: 87–100, 10.1016/S1381-1177(03)00075-4, 1:CAS:528:DC%2BD3sXms12itrw%3DCrossRefGoogle Scholar
  2. 2.
    Krebs H A. Metabolism of amino-acids: The synthesis of glutamine from glutamate and ammonia, and the enzymatic hydrolysis of glutamine in animal tissues. Biochem J, 1935, 29: 1951–1969, 16745865, 1:CAS:528:DyaA2MXmtFSrug%3D%3DCrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Watford M, Smith E M, Erbelding E J. The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat. Biochem J, 1984, 224: 207–214, 6508757, 1:CAS:528:DyaL2MXktFShCrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Smith E M, Watford M. Molecular cloning of a cDNA for rat hepatic glutaminase: Sequence similarity to kidney-type glutaminase. J Biol Chem, 1990, 265: 10631–10636, 2191954, 1:CAS:528:DyaK3cXkvFeqt78%3DPubMedGoogle Scholar
  5. 5.
    Curthoys N P, Watford M. Regulation of glutaminase activity and glutamine metabolism. Annu Rev Nutr, 1995, 15: 133–159, 8527215, 10.1146/annurev.nu.15.070195.001025, 1:CAS:528:DyaK2MXntVCjsro%3DCrossRefPubMedGoogle Scholar
  6. 6.
    Masola B, Zvinavashe E. Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions. Amino Acids, 2003, 24: 427–434, 12768506, 10.1007/s00726-002-0312-x, 1:CAS:528:DC%2BD3sXntlaltbg%3DCrossRefPubMedGoogle Scholar
  7. 7.
    Márquez J, López de la Oliva A R, Matés M J, et al. Glutaminase: A multifaceted protein not only involved in generating glutamate. Neurochem Int, 2006, 48: 465–471, 16516349CrossRefPubMedGoogle Scholar
  8. 8.
    Prusiner S. Glutaminases of Escherichia coli: Properties, regulation and evolution. In: Prusiner S, Stadtman E R, eds. The Enzymes of Glutamine Metabolism. New York: Academic Press, 1973. 293–316Google Scholar
  9. 9.
    Prusiner S, Davis J N, Stadtman E R. Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability? J Biol Chem, 1976, 251: 3447–3456, 6454, 1:CAS:528:DyaE28XksVaktrw%3DPubMedGoogle Scholar
  10. 10.
    Duran S, Sanchez-Linares L, Huerta-Saquero A, et al. Identification of two glutaminases in Rhizobium etli. Biochem Genet, 1996, 34: 453–465, 9126674, 10.1007/BF00570126, 1:CAS:528:DyaK2sXisl2qu7s%3DCrossRefPubMedGoogle Scholar
  11. 11.
    Masuo N, Ito K, Yoshimune K, et al. Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40. Protein Expres Purif, 2004, 38: 272–278, 10.1016/j.pep.2004.09.003, 1:CAS:528:DC%2BD2cXhtVSnt7jECrossRefGoogle Scholar
  12. 12.
    Wakayama M, Yamagata T, Kamemura A, et al. Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation. J Ind Microbiol Biotechnol, 2005, 32: 383–390, 16012776, 10.1007/s10295-005-0257-7, 1:CAS:528:DC%2BD2MXht1WntLjLCrossRefPubMedGoogle Scholar
  13. 13.
    Kaneko T, Sato S, Kotani H, et al. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res, 1996, 3: 109–136, 8905231, 10.1093/dnares/3.3.109, 1:CAS:528:DyaK28Xmtl2qsLc%3DCrossRefPubMedGoogle Scholar
  14. 14.
    Muro-Pastor M I, Reyes J C, Florencio F J. Ammonium assimilation in cyanobacteria. Photosynth Res, 2005, 83: 135–150, 16143848, 10.1007/s11120-004-2082-7, 1:CAS:528:DC%2BD2MXhslGntrg%3DCrossRefPubMedGoogle Scholar
  15. 15.
    Chen C H, Baalen C V, Tabita F R. DL-7-azatryptophan and citrulline metabolism in the cyanobacterium Anabaena sp. strain 1F. J Bacteriol, 1987, 169: 1114–1119, 2880834, 1:CAS:528:DyaL2sXhsFaktLw%3DPubMedCentralPubMedGoogle Scholar
  16. 16.
    Allen M M. Simple conditions for growth of unicellular blue-green algae on plates. J Phycol, 1968, 4: 1–4, 10.1111/j.1529-8817.1968.tb04667.x, 1:CAS:528:DyaF1MXotVKlCrossRefGoogle Scholar
  17. 17.
    Thompson J D, Higgins D G, Gibson T J. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 1994, 22(Suppl 22): 4673–4680, 7984417, 10.1093/nar/22.22.4673, 1:CAS:528:DyaK2MXitlSgu74%3DCrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Williams J G K. Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol, 1988, 167: 766–778, 10.1016/0076-6879(88)67088-1, 1:CAS:528:DyaL1MXhvVKqs7g%3DCrossRefGoogle Scholar
  19. 19.
    Kenny J, Bao Y, Hamm B, et al. Bacterial expression, purification, and characterization of rat kidney-type mitochondrial glutaminase. Protein Expr Purif, 2003, 31: 140–148, 12963351, 10.1016/S1046-5928(03)00161-X, 1:CAS:528:DC%2BD3sXntVGkt7s%3DCrossRefPubMedGoogle Scholar
  20. 20.
    Cann M J, Hammer A, Zhou J, et al. A defined subset of adenylyl cyclases is regulated by bicarbonate ion. J Biol Chem, 2003, 278: 35033–35038, 12829712, 10.1074/jbc.M303025200, 1:CAS:528:DC%2BD3sXntVWqt74%3DCrossRefPubMedGoogle Scholar
  21. 21.
    Curthoys N P, Weiss R F. Regulation of renal ammoniagenesis. Subcellular localization of rat kidney glutaminase isoenzymes. J Biol Chem, 1974, 249: 3261–3266, 4364420, 1:CAS:528:DyaE2cXkt1entr8%3DPubMedGoogle Scholar
  22. 22.
    Oka A, Sugisake H, Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn 903. J Mol Biol, 1981, 147: 217–226, 6270337, 10.1016/0022-2836(81)90438-1, 1:CAS:528:DyaL3MXkslGltro%3DCrossRefPubMedGoogle Scholar
  23. 23.
    Wang H L, Postier B L, Burnap R L. Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803. Mol Microbiol, 2002, 44: 1493–1506, 12067339, 10.1046/j.1365-2958.2002.02983.x, 1:CAS:528:DC%2BD38Xlt1amtrw%3DCrossRefPubMedGoogle Scholar
  24. 24.
    Porra R J, Thompson W A, Kriedemann P E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta, 1989, 975: 384–394, 10.1016/S0005-2728(89)80347-0, 1:CAS:528:DyaL1MXkvFehtL4%3DCrossRefGoogle Scholar
  25. 25.
    Vioque A. Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. Nucleic Acids Res, 1992, 20: 6331–6337, 1282240, 10.1093/nar/20.23.6331, 1:CAS:528:DyaK3sXit1Ojsr4%3DCrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Yano S, Kamemura A, Yoshimune K, et al. Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3. J Biosci Bioeng, 2006, 102: 362–364, 17116585, 10.1263/jbb.102.362, 1:CAS:528:DC%2BD28Xhtlemsr3PCrossRefPubMedGoogle Scholar
  27. 27.
    Klein M, Kaltwasser H, Thomas J. Isolation of a novel, phosphate-activated glutaminase from Bacillus pasteurii. FEMS Microbiol Lett, 2002, 206: 63–67, 11786258, 10.1111/j.1574-6968.2002.tb10987.x, 1:CAS:528:DC%2BD38XitFahsg%3D%3DCrossRefPubMedGoogle Scholar
  28. 28.
    Allakhverdiev S I, Sakamoto A, Nishiyama Y, et al. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp. Plant Physiol, 2000, 123: 1047–1056, 10889254, 10.1104/pp.123.3.1047, 1:CAS:528:DC%2BD3cXlt1Slt74%3DCrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Kanesaki Y, Suzuki I, Allakhverdiev S I, et al. Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803. Biochem Biophys Res Commun, 2002, 290: 339–348, 11779175, 10.1006/bbrc.2001.6201, 1:CAS:528:DC%2BD38XhsFCnuw%3D%3DCrossRefPubMedGoogle Scholar
  30. 30.
    Muro-Pastor M I, Florencio F J. Regulation of ammonium assimilation in cyanobacteria. Plant Physiol Biochem, 2003, 41: 595–603, 10.1016/S0981-9428(03)00066-4, 1:CAS:528:DC%2BD3sXlsVals78%3DCrossRefGoogle Scholar
  31. 31.
    Moriguchi M, Sakai K, Tateyama R, et al. Isolation and characterization of salt-tolerant glutaminase from marine Micrococcus luteus K-3. J Ferment Bioeng, 1994, 77: 621–625, 10.1016/0922-338X(94)90143-0, 1:CAS:528:DyaK2cXlslemt7s%3DCrossRefGoogle Scholar
  32. 32.
    Marin K, Suzuki I, Yamaguchi K, et al. Identification of histidine kinases that act as sensors in the perception of salt stress in Synechocystis sp. PCC 6803. Proc Natl Acad Sci USA, 2003, 100: 9061–9066, 12853569, 10.1073/pnas.1532302100, 1:CAS:528:DC%2BD3sXlvVyhsr4%3DCrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Huang F, Fulda S, Hagemann M, et al. Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803. Proteomics, 2006, 6: 910–920, 16400685, 10.1002/pmic.200500114, 1:CAS:528:DC%2BD28XhvFWku7o%3DCrossRefPubMedGoogle Scholar
  34. 34.
    Fulda S, Mikkat S, Huang F, et al. Proteome analysis of salt stress response in the cyanobacterium Synechocystis sp. strain PCC 6803. Proteomics, 2006, 6: 2733–2745, 16572470, 10.1002/pmic.200500538, 1:CAS:528:DC%2BD28XltF2it7s%3DCrossRefPubMedGoogle Scholar

Copyright information

© Science in China Press and Springer-Verlag GmbH 2008

Authors and Affiliations

  • Jie Zhou
    • 1
  • JunXia Zhou
    • 1
    • 2
  • HaoMeng Yang
    • 1
  • ChengShi Yan
    • 1
  • Fang Huang
    • 1
  1. 1.Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of SciencesBeijingChina
  2. 2.Graduate School of Chinese Academy of SciencesBeijingChina

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