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glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II

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Abstract

The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis–Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.

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Abbreviations

TB:

Tuberculosis

FBPase:

Fructose 1,6–bisphosphatase

Ni– NTA:

Nickel-nitrilotriacetic acid

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

kDa:

Kilodalton

IPTG:

Isopropyl-b-D-thiogalactopyranoside

FPLC:

Fast performance liquid chromatography

SEC:

Size exclusion chromatography

NADP+ :

Nicotinamide adenine dinucleotide phosphate

NADPH:

Nicotinamide adenine dinucleotide phosphate reduced form

DTT:

Dithiothreitol

PGI:

Phosphoglucoisomerase

G6PDH:

Glucose-6-phosphate dehydrogenase

References

  1. WHO, World Health Organization (2008) Global tuberculosis control: surveillance, planning, financing. WHO Report.

  2. Russell, D. G., Cardona, P. J., Kim, M. J., Allain, S., & Altare, F. (2009). Nature Immunology, 10, 943–948.

    Article  CAS  Google Scholar 

  3. Boshoff, H. I., & Barry, C. E., 3rd. (2005). Nature Reviews. Microbiology, 3, 70–80.

    Article  CAS  Google Scholar 

  4. McKinney, J. D., Honer zu Bentrup, K., Munoz-Elias, E. J., Miczak, A., Chen, B., Chan, W. T., et al. (2000). Nature, 406, 735–738.

    Article  CAS  Google Scholar 

  5. Munoz-Elias, E. J., & McKinney, J. D. (2005). Natural Medicines, 11, 638–644.

    Article  CAS  Google Scholar 

  6. Dunn, M. F., Ramirez-Trujillo, J. A., & Hernandez-Lucas, I. (2009). Microbiology, 155, 3166–3175.

    Article  CAS  Google Scholar 

  7. Sharma, V., Sharma, S., Hoener zu Bentrup, K., McKinney, J. D., Russell, D. G., Jacobs, W. R., Jr., et al. (2000). Nature Structural Biology, 7, 663–668.

    Article  CAS  Google Scholar 

  8. Smith, C. V., Huang, C. C., Miczak, A., Russell, D. G., Sacchettini, J. C., & Honer zu Bentrup, K. (2003). The Journal of Biological Chemistry, 278, 1735–1743.

    Article  CAS  Google Scholar 

  9. Anstrom, D. M., & Remington, S. J. (2006). Protein Science, 15, 2002–2007.

    Article  CAS  Google Scholar 

  10. Purohit, H. J., Cheema, S., Lal, S., Raut, C. P., & Kalia, V. C. (2007). Infectious Disorders Drug Targets, 7, 245–250.

    Article  CAS  Google Scholar 

  11. Liu, K., Yu, J., & Russell, D. G. (2003). Microbiology, 149, 1829–1835.

    Article  CAS  Google Scholar 

  12. Marrero, J., Rhee, K. Y., Schnappinger, D., Pethe, K., & Ehrt, S. (2010). Proceedings of the National Academy of Sciences of the United States of America, 107, 9819–9824.

    Article  CAS  Google Scholar 

  13. Movahedzadeh, F., Rison, S. C., Wheeler, P. R., Kendall, S. L., Larson, T. J., & Stoker, N. G. (2004). Microbiology, 150, 3499–3505.

    Article  CAS  Google Scholar 

  14. Donahue, J. L., Bownas, J. L., Niehaus, W. G., & Larson, T. J. (2000). Journal of Bacteriology, 182, 5624–5627.

    Article  CAS  Google Scholar 

  15. Nishimasu, H., Fushinobu, S., Shoun, H., & Wakagi, T. (2004). Structure, 12, 949–959.

    Article  CAS  Google Scholar 

  16. Hines, J. K., Fromm, H. J., & Honzatko, R. B. (2006). The Journal of Biological Chemistry, 281, 18386–18393.

    Article  CAS  Google Scholar 

  17. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., et al. (1998). Nature, 393, 537–544.

    Article  CAS  Google Scholar 

  18. Brown, G., Singer, A., Lunin, V. V., Proudfoot, M., Skarina, T., Flick, R., et al. (2009). The Journal of Biological Chemistry, 284, 3784–3792.

    Article  CAS  Google Scholar 

  19. Rittmann, D., Schaffer, S., Wendisch, V. F., & Sahm, H. (2003). Archives of Microbiology, 180, 285–292.

    Article  CAS  Google Scholar 

  20. Sassetti, C. M., & Rubin, E. J. (2003). Proceedings of the National Academy of Sciences of the United States of America, 100, 12989–12994.

    Article  CAS  Google Scholar 

  21. York, J. D., Ponder, J. W., & Majerus, P. W. (1995). Proceedings of the National Academy of Sciences of the United States of America, 92, 5149–5153.

    Article  CAS  Google Scholar 

  22. Patel, S., Martinez-Ripoll, M., Blundell, T. L., & Albert, A. (2002). Journal of Molecular Biology, 320, 1087–1094.

    Article  CAS  Google Scholar 

  23. Albert, A., Yenush, L., Gil-Mascarell, M. R., Rodriguez, P. L., Patel, S., Martinez-Ripoll, M., et al. (2000). Journal of Molecular Biology, 295, 927–938.

    Article  CAS  Google Scholar 

  24. McAllister, G., Whiting, P., Hammond, E. A., Knowles, M. R., Atack, J. R., Bailey, F. J., et al. (1992). The Biochemical Journal, 284(Pt 3), 749–754.

    CAS  Google Scholar 

  25. Nigou, J., Dover, L. G., & Besra, G. S. (2002). Biochemistry, 41, 4392–4398.

    Article  CAS  Google Scholar 

  26. Chen, L., & Roberts, M. F. (2000). Biochemistry, 39, 4145–4153.

    Article  CAS  Google Scholar 

  27. Peng, Z., & Verma, D. P. (1995). The Journal of Biological Chemistry, 270, 29105–29110.

    Article  CAS  Google Scholar 

  28. Chen, L., & Roberts, M. F. (1999). Applied and Environmental Microbiology, 65, 4559–4567.

    CAS  Google Scholar 

  29. Chen, L., & Roberts, M. F. (1998). Applied and Environmental Microbiology, 64, 2609–2615.

    CAS  Google Scholar 

  30. Gu, X., Chen, M., Shen, H., Jiang, X., Huang, Y., & Wang, H. (2006). Biochemical and Biophysical Research Communications, 339, 897–904.

    Article  CAS  Google Scholar 

  31. Kelley-Loughnane, N., Biolsi, S. A., Gibson, K. M., Lu, G., Hehir, M. J., Phelan, P., et al. (2002). Biochimica et Biophysica Acta, 1594, 6–16.

    Article  CAS  Google Scholar 

  32. Hines, J. K., Chen, X., Nix, J. C., Fromm, H. J., & Honzatko, R. B. (2007). The Journal of Biological Chemistry, 282, 36121–36131.

    Article  CAS  Google Scholar 

  33. Hines, J. K., Fromm, H. J., & Honzatko, R. B. (2007). The Journal of Biological Chemistry, 282, 11696–11704.

    Article  CAS  Google Scholar 

  34. Hines, J. K., Kruesel, C. E., Fromm, H. J., & Honzatko, R. B. (2007). The Journal of Biological Chemistry, 282, 24697–24706.

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to acknowledge Maxwell Rutter for technical assistance with experimental work and proof reading of the manuscript; Prof. Michael Johnson, Dr. Cele Abad-Zapatero and Dr. Shahila Mehboob for providing facility, equipment and technical assistance to perform size exclusion chromatography for protein purification; Prof. Chuan He and Pedro Brugarolas for providing facility, equipment and technical assistance to perform molecular weight estimation of the purified protein. The authors are thankful to American Lung Association (Grant No. RG-82534-N).

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Authors and Affiliations

  1. Institute for Tuberculosis Research (M/C 964), College of Pharmacy, Room 412, University of Illinois at Chicago, 833 S. Wood St, Chicago, IL, 60612–7231, USA

    Hiten J. Gutka, Kamolchanok Rukseree, Scott G. Franzblau & Farahnaz Movahedzadeh

  2. Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL, 60607, USA

    Hiten J. Gutka, Scott G. Franzblau & Farahnaz Movahedzadeh

  3. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Pathumthani, 12120, Thailand

    Kamolchanok Rukseree

  4. Veterinary Laboratory Agency, Weybridge, New Haw, Addlestone, KT15 3NB, Surrey, UK

    Paul R. Wheeler

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  1. Hiten J. Gutka
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  2. Kamolchanok Rukseree
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  3. Paul R. Wheeler
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  4. Scott G. Franzblau
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  5. Farahnaz Movahedzadeh
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Correspondence to Farahnaz Movahedzadeh.

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Gutka, H.J., Rukseree, K., Wheeler, P.R. et al. glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II. Appl Biochem Biotechnol 164, 1376–1389 (2011). https://doi.org/10.1007/s12010-011-9219-x

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  • DOI: https://doi.org/10.1007/s12010-011-9219-x

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