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Evidence of the Involvement of Asparagine Deamidation in the Formation of Cyclodextrin Glycosyltransferase Isoforms in Paenibacillus sp. RB01

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Abstract

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.

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Abbreviations

CGTase:

Cyclodextrin glycosyltransferase

CD:

Cyclodextrins

A11:

Paenibacillus sp. A11

RB01:

Paenibacillus sp. RB01

CD value:

Coefficient of deamidation

isoAsp:

Isoaspartate

N/Asn:

Asparagine

D/Asp:

Aspartic acid

References

  1. van der Veen, A. B., van Alebeek, G. J. W. M., Uitdehaag, J. C. M., Dijkstra, B. W., & Dijkhuizen, L. (2000). The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms. European Journal of Biochemistry, 267, 658–665.

    Article  Google Scholar 

  2. Qi, Q., & Zimmermann, W. (2005). Cyclodextrin glucanotransferase: From gene to applications. Applied Microbiology and Biotechnology, 66, 475–485.

    Article  CAS  Google Scholar 

  3. Wang, F., Du, C. G., Li, Y., & Chen, J. (2004). Optimization of cultivation conditions for the production of γ-cyclodextrin glucanotransferase by Bacillus macorous. Food Biotechnology, 18, 251–264.

    Article  CAS  Google Scholar 

  4. Hashimoto, H. (2002). Present status of industrial application of cyclodextrins in Japan. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 57–62.

    Article  CAS  Google Scholar 

  5. Chittiteeranon, P., Soontaros, S., & Pongsawasdi, P. (2007). Preparation and characterization of inclusion complexes containing fixolide, a synthetic musk fragrance and cyclodextrins. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 57, 69–71.

    Article  CAS  Google Scholar 

  6. [6] Tesana, S. (2001). Cyclodextrin glycosyltransferase from thermotolerant bacteria: screening, optimization, partial purification and characterization. MSc. Thesis, Chulalongkorn University, Bangkok, Thailand.

  7. Yenpetch, W., Kaulpiboon, J., Iizuka, M., & Pongsawasdi, P. (2004). Thermostable CGTase from Paenibacillus sp. RB01 and its chemical modification with glucomannan. Proceedings of the 12th International Cyclodextrin Symposium (pp. 107–112). Montpellier, France.

  8. Abelyan, V. A., Yamamoto, T., & Afrikyan, E. G. (1994). Isolation and characterization of cyclodextrin glucanotransferase using cyclodextrin polymers and their derivatives. Biochemistry (Moscow), 59, 573–579.

    Google Scholar 

  9. Volkova, D. A., Lopatin, S. A., Gracheva, I. M., & Varlamov, V. P. (2001). Preparation of high purity cyclodextrin glucanotransferase from Bacillus sp. 1070. Applied Biochemistry and Microbiology, 37(2), 138–141.

    Article  CAS  Google Scholar 

  10. Rojtinnakorn, J., Kim, P., Laloknam, S., Tongsima, A., Kamolsiripichaiporn, S., Limpaseni, T., et al. (2001). Immunoaffinity purification and characterization of cyclodextrin glycosyltransferase from Bacillus circulans A11. Science Asia, 27, 105–112.

    Article  CAS  Google Scholar 

  11. Prasong, W. (2002). Structural analysis of cyclodextrin glycosyltransferase isoforms from Paenibacillus sp. A11. MSc Thesis, Chulalongkorn University, Bangkok, Thailand.

  12. Robinson, N. E., & Robinson, A. B. (2001). Molecular clocks. Proceedings of the National Academy of Sciences, 98, 944–949.

    Article  CAS  Google Scholar 

  13. Robinson, N. E. (2002). Protein deamidation. Proceedings of the National Academy of Sciences, 99, 5283–5288.

    Article  CAS  Google Scholar 

  14. Bischoff, R., & Kolbe, H. (1994). Deamidation of asparagine and glutamine residues in proteins and peptides: Structural determinants and analytical methodology. Journal of Chromatography B, 662, 261–278.

    Article  CAS  Google Scholar 

  15. Flatmark, T., & Sletten, K. (1968). Multiple forms of cytochrome c in the rat precursor-product relationship between the main component Cy I and the minor components Cy II and Cy III in vivo. Journal of Biological Chemistry, 243, 1623–1629.

    CAS  Google Scholar 

  16. Robinson, N. E., & Robinson, A. B. (2001). Prediction of protein deamidation rates from primary and three-dimensional structure. Proceedings of the National Academy of Sciences, 98, 4367–4372.

    Article  CAS  Google Scholar 

  17. Zomber, G., Reuveny, S., Garti, N., Shafferman, A., & Elhanany, E. (2005). Effects of spontaneous deamidation on cytotoxic activity of the Bacillus anthracis protective antigen. Journal of Biological Chemistry, 280, 39897–39906.

    Article  CAS  Google Scholar 

  18. Cox, G. A., Johnson, R. B., Cook, J. A., Wakulchik, M., Johnson, M. G., Villarreal, E. V., et al. (1999). Identification and characterization of human rhinovirus-14 3C protease deamidation isoform. Journal of Biological Chemistry, 274, 13211–13216.

    Article  CAS  Google Scholar 

  19. Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., & Flatmark, T. (2003). Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase structural and functional implications. European Journal of Biochemistry, 270, 929–938.

    Article  CAS  Google Scholar 

  20. Goel, A., & Nene, N. S. (1995). Modifications in the phenolphthalein method for spectrophotometric estimation of beta cyclodextrin. Starch/Starke, 47, 399–400.

    Article  CAS  Google Scholar 

  21. Ellis, K. J., & Morrison, J. F. (1982). Buffers of constant ionic strength for studying pH-dependent processes. Methods in Enzymology, 87, 405–426.

    Article  CAS  Google Scholar 

  22. Bovetto, L. J., Backe, D. P., Villette, J. R., Sicard, P. J., & Bouquelet, S. J.-L. (1992). Cyclomaltodextrin glucanotransferase from Bacillus circulans E192 I : Purification and characterization of the enzyme. Biotechnology and Applied Biochemistry, 15, 48–58.

    CAS  Google Scholar 

  23. Wind, R. D., Liebl, W., Buitellaar, R. M., Penninga, D., Spreinat, A., Dijkhuizen, L., et al. (1995). Cyclodextrin formation by the thermostable α-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase. Applied and Environmental Microbiology, 61, 1257–1265.

    CAS  Google Scholar 

  24. Peters, B., & Trout, B. L. (2006). Asparagine deamidation: pH dependence mechanism from density functional theory. Biochemistry, 45, 5384–5392.

    Article  CAS  Google Scholar 

  25. Geiger, T., & Clarck, S. (1987). Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides-succinimide-linked reactions that contribute to protein degradation. Journal of Biological Chemistry, 262, 785–794.

    CAS  Google Scholar 

  26. DeLuna, A., Quezada, H., Gomez-Puyou, A., & Gonzalez, A. (2005). Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae. Biochemical and Biophysical Research Communications, 328, 1083–1090.

    Article  CAS  Google Scholar 

  27. Flatmark, T., & Vesterberg, O. (1966). On the heterogeneity of beef heart cytochrome c IV Isoelectric fractionation by electrolysis in a natural pH gradient. Acta Chemica Scandinavica, 20, 1497–1503.

    Article  CAS  Google Scholar 

  28. Kaskangam, K. (1998). Isolation and characterization of cyclodextrin glycosyltransferase isozymes from Bacillus sp. A11. MSc Thesis, Chulalongkorn University, Bangkok, Thailand.

  29. Zhang, W., & Czupryn, M. J. (2003). Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms. Journal of Pharmaceutical Biomedical Analysis, 30, 1479–1490.

    Article  CAS  Google Scholar 

  30. Perkins, M., Theiler, R., Lunte, S., & Jeschke, M. (2000). Determination of the origin of charge heterogeneity in a murine monoclonal antibody. Pharmaceutical Research, 17, 1110–1117.

    Article  CAS  Google Scholar 

  31. Curnis, F., Longhi, R., Crippa, L., Cattaneo, A., Dondossola, E., Bachi, A., et al. (2006). Spontaneous formation of l-isoaspartate and gain of function in fibronectin. Journal of Biological Chemistry, 281, 36466–36476.

    Article  CAS  Google Scholar 

  32. Kimura, K., Kataoka, S., Ishii, Y., Takano, T., & Yamane, K. (1987). Isoaspartate in ribosomal protein S11 of Escherichia coli. Journal of Bacteriology, 169, 4399–4402.

    CAS  Google Scholar 

  33. Yenpetch, W., Packdibumrung, K., Zimmermann, W., & Pongsawasdi. P (2010) Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase. Journal of Inclusion Phenomena and Macrocyclic Chemistry. doi:10.1007/s10847-010-9856-7.

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Acknowledgments

WY was supported by a RGJ PhD Fellowship from the Thailand Research Fund. Financial supports from the Rachadapiseksompote Endowment Fund and from the 90th Anniversary of Chulalongkorn University Fund are acknowledged. We also acknowledge the support from the Thai Government Stimulus Package 2 (TKK2555) under the Project PERFECTA. Special thanks go to Dr. Robert Butcher of the Publication Counseling Unit of the Faculty of Science for editing the manuscript.

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Authors and Affiliations

  1. Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Wanchai Yenpetch, Kanoktip Packdibamrung & Piamsook Pongsawasdi

  2. Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig, Germany

    Wolfgang Zimmermann

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  1. Wanchai Yenpetch
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  2. Kanoktip Packdibamrung
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  3. Wolfgang Zimmermann
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  4. Piamsook Pongsawasdi
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Correspondence to Piamsook Pongsawasdi.

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Yenpetch, W., Packdibamrung, K., Zimmermann, W. et al. Evidence of the Involvement of Asparagine Deamidation in the Formation of Cyclodextrin Glycosyltransferase Isoforms in Paenibacillus sp. RB01. Mol Biotechnol 47, 234–242 (2011). https://doi.org/10.1007/s12033-010-9337-7

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