Skip to main content
SpringerLink
Log in

Recombinant Deamidated Mutants of Erwinia chrysanthemi l-Asparaginase Have Similar or Increased Activity Compared to Wild-Type Enzyme

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The enzyme Erwinia chrysanthemi l-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s−1) when compared to the WT (k cat 565 s−1), which was further increased in the double mutant, with a k cat of 798 s−1. Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Beard, M. E. J., Crowther, D., Galton, D. A. G., Guyer, R. J., Fairley, G. H., Kay, H. E. M., et al. (1970). l-Asparaginase in treatment of acute leukaemia and lymphosarcoma. British Medical Journal, 1, 191–195.

    Article  CAS  Google Scholar 

  2. Pieters, R., Hunger, S. P., Boos, J., Rizzari, C., Silverman, L., Baruchel, A., et al. (2011). l-Asparaginase treatment in acute lymphoblastic leukemia: A focus on Erwinia asparaginase. Cancer, 117(2), 238–249.

    Article  CAS  Google Scholar 

  3. Vrooman, L. M., Supko, J. G., Neuberg, D. S., Asselin, B. L., Athale, U. H., Clavell, L., et al. (2010). Erwinia asparaginase after allergy to E. coli asparaginase in children with acute lymphoblastic leukemia. Pediatric Blood & Cancer, 54(2), 199–205.

    Google Scholar 

  4. Broome, J. D. (1968). Factors which may influence the effectiveness of l-asparaginases as tumor inhibitors. British Journal of Cancer, 22(3), 595–602.

    Article  CAS  Google Scholar 

  5. Wriston, J. C. (1985). Asparaginase. Methods in Enzymology, 113, 608–618.

    Article  CAS  Google Scholar 

  6. Aghaiypour, K., Wlodawer, A., & Lubkowski, J. (2001). Structural basis for the activity and substrate specificity of Erwinia chrysanthemi l-asparaginase. Biochemistry, 40, 5655–5664.

    Article  CAS  Google Scholar 

  7. Lubkowski, J., Dauter, M., Aghaiypour, K., Wlodawer, A., & Dauter, Z. (2003). Atomic resolution structure of Erwinia chrysanthemi l-asparaginase. Acta Crystallographica Section D: Biological Crystallography, 59, 84–92.

    Article  Google Scholar 

  8. Miller, M., Rao, J. K. M., Wlodawer, A., & Gribskov, M. R. (1993). A left-handed crossover involved in amidohydrolase catalysis: Crystal structure of Erwinia chrysanthemi l-asparaginase with bound l-aspartate. FEBS Journal, 328(3), 275–279.

    Article  CAS  Google Scholar 

  9. Gervais, D., Allison, N., Jennings, A., Jones, S., & Marks, T. (2013). Validation of a thirty-year-old process for the manufacture of l-asparaginase from erwinia chrysanthemi. Bioprocess and Biosystems Engineering, 36(4), 453–460.

    Article  CAS  Google Scholar 

  10. Aswad, D. W. (1995). Deamidation and isoaspartate formation in peptides and proteins. Boca Raton, FL: CRC Press.

    Google Scholar 

  11. Aswad, D. W., Paranandi, M. V., & Schurter, B. T. (2000). Isoaspartate in peptides and proteins: formation, significance and analysis. Journal of Pharmaceutical and Biomedical Analysis, 21, 1129–1136.

    Article  CAS  Google Scholar 

  12. Gervais, D., O’Donnell, J., Sung, M., & Smith, S. (2013). Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase. Process Biochemistry, 48(9), 1311–1316.

    Article  CAS  Google Scholar 

  13. Zhang, T., Bourret, J., & Cano, T. (2011). Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography. J Chromatography A, 1218(31), 5079–5086.

    Article  CAS  Google Scholar 

  14. Khawli, L. A., Goswami, S., Hutchinson, R., Kwong, Z. W., Yang, J., Wang, X., et al. (2010). Charge variants in IgG1 isolation, characterization, in vitro binding properties and pharmacokinetics in rats. MAbs, 2(6), 613–624.

    Article  Google Scholar 

  15. Laboureur, P., Langlois, C., Labrousse, M., Boudon, M., Emeraud, J., Samain, J. F., et al. (1971). l-Asparaginases from Escherichia coli. I. Properties of the native forms. Biochimie, 53, 1147–1156.

    Article  CAS  Google Scholar 

  16. Laboureur, P., Langlois, C., Labrousse, M., Boudon, M., Emeraud, J., Samain, J. S., et al. (1971). l-Asparaginases d’Escherichia coli II: Plurality and origin of molecular forms: Relations with the biological activity. Biochimie, 53, 1157–1165.

    Article  CAS  Google Scholar 

  17. Wagner, O., Irion, E., Arens, A., & Bauer, K. (1969). Partially deaminated l-asparaginase. Biochemical and Biophysical Research Communications, 37(3), 383–392.

    Article  CAS  Google Scholar 

  18. Bae, N., Pollak, A., & Lubec, G. (2011). Proteins from Erwinia asparaginase Erwinase® and E. coli asparaginase 2 MEDAC® for treatment of human leukaemia, show a multitude of modifications for which the consequences are completely unclear. Electrophoresis, 32, 1824–1828.

    Article  CAS  Google Scholar 

  19. Gupta, R., & Srivastava, O. P. (2004). Effect of deamidation of asparagine 146 on functional and structural properties of human lens αB-crystallin. Investigative Ophthalmology & Visual Science, 45(1), 206–214.

    Article  Google Scholar 

  20. Takata, T., Oxford, J. T., Demeler, B., & Lampi, K. J. (2008). Deamidation destabilises and triggers aggregation of a lens protein, βA3-crystallin. Protein Science, 17, 1565–1575.

    Article  CAS  Google Scholar 

  21. Minton, N. P., Bullman, H. M. S., Scawen, M. D., Atkinson, T., & Gilbert, H. J. (1986). Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 l-asparaginase gene. Gene, 46, 25–35.

    Article  CAS  Google Scholar 

  22. Sadler, J. R., Sasmor, H., & Betz, J. L. (1983). A perfectly symmetric lac operator binds the lac repressor very tightly. Proceedings of the National Academy of Sciences of the United States of America, 80(22), 6785–6789.

    Article  CAS  Google Scholar 

  23. Oehler, S., Amouyal, M., Kolkhof, P., von Wilkcken-Bergmann, B., & Müller-Hill, B. (1994). Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO Journal, 13(14), 3348–3355.

    CAS  Google Scholar 

  24. Gentz, R., & Bujard, H. (1985). Promoters recognised by Escherichia coli RNA polymerase selected by function: Highly efficient promoters from bacteriophage T5. Journal of Bacteriology, 164(1), 70–77.

    CAS  Google Scholar 

  25. Lanzer, M., & Bujard, H. (1988). Promoters largely determine the efficiency of repressor action. Proceedings of the National Academy of Sciences of the United States of America, 85(23), 8973–8977.

    Article  CAS  Google Scholar 

  26. Kotzia, G. A., & Labrou, N. E. (2005). Cloning, expression and characterisation of Erwinia carotovora l-asparaginase. Journal of Biotechnology, 119, 309–323.

    Article  CAS  Google Scholar 

  27. Lee, S. M., Wroble, M. H., & Ross, J. T. (1989). l-Asparaginase from Erwinia carotovora: An improved recovery and purification process using affinity chromatography. Applied Biochemistry and Biotechnology, 22(1), 1–11.

    Article  CAS  Google Scholar 

  28. Lowry, O. H., Rosbrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  29. Harms, E., Wehner, A., Jennings, M. P., Pugh, K. J., Beacham, I. R., & Röhm, K. H. (1991). Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts. Protein Expression and Purification, 2, 144–150.

    Article  CAS  Google Scholar 

  30. Papageorgiou, A. C., Posypanova, G. A., Andersson, C. S., Sokolov, N. N., & Krasotkina, J. (2008). Structural and functional insights into Erwinia carotovora l-asparaginase. FEBS Journal, 275, 4306–4316.

    Article  CAS  Google Scholar 

  31. Whitmore, L., & Wallace, B. A. (2008). Protein Secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers, 89, 392–400.

    Article  CAS  Google Scholar 

  32. Whitmore, L., & Wallace, B. A. (2004). DICHROWEB: An online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Research, 32, W668–W673.

    Article  CAS  Google Scholar 

  33. Lobley, A., Whitmore, L., & Wallace, B. A. (2002). DICHROWEB: An interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics, 18, 211–212.

    Article  CAS  Google Scholar 

  34. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., et al. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7, 539.

    Article  Google Scholar 

  35. Gouet, P., Robert, X., & Courcelle, E. (2003). ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Research, 31(13), 3320–3323.

    Article  CAS  Google Scholar 

  36. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., et al. (2004). UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605–1612.

    Article  CAS  Google Scholar 

  37. Buck, P. W., Elsworth, R., Miller, G. A., Sargeant, K., Stanley, J. L., & Wade, H. E. (1971). The batch production of l-asparaginase from Erwinia carotovora. Journal of General Microbiology, 65, i.

    Article  CAS  Google Scholar 

  38. Tyler-Cross, R., & Schirch, V. (1991). Effects of amino acid sequence, buffer, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. Journal of Biological Chemistry, 266(33), 22549–22556.

    CAS  Google Scholar 

  39. Xie, M., & Schowen, R. L. (1999). Secondary structure and protein deamidation. Journal of Pharmaceutical Sciences, 88(1), 8–13.

    Article  CAS  Google Scholar 

  40. Patel, K., & Borchardt, R. T. (1990). Chemical pathways of peptide degradation. II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide. Pharmaceutical Research, 7, 703–711.

    Article  CAS  Google Scholar 

  41. Lascu, I., Chaffotte, A., Limbourg-Bouchon, B., & Veron, M. (1992). A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme. Journal of Biological Chemistry, 267, 12775–12781.

    CAS  Google Scholar 

  42. Ashrafi, H., Amini, M., Mohammadi-Samani, S., Ghasemi, Y., Azadi, A., Tabandeh, M. R., et al. (2013). Nanostructure l-asparaginase-fatty acid bioconjugate: Synthesis, preformulation study and biological assessment. International Journal of Biological Macromolecules, 62, 180–187.

    Article  CAS  Google Scholar 

  43. Jameel, F., Bogner, R., Mauri, F., & Kalonia, D. (1997). Investigation of physicochemical changes to l-asparaginase during freeze-thaw cycling. Journal of Pharmacy and Pharmacology, 49, 472–477.

    Article  CAS  Google Scholar 

  44. Mills, J. E., & Dean, P. M. (1996). Three-dimensional hydrogen-bond geometry and probability information from a crystal survey. Journal of Computer-Aided Molecular Design, 10(6), 607–622.

    Article  Google Scholar 

  45. Dunbrack, R. L. (2002). Rotamer libraries in the 21st century. Current Opinion in Structural Biology, 12(4), 431–440.

    Article  CAS  Google Scholar 

  46. Krasotkina, J., Borisova, A. A., Gervaziev, Y. V., & Sokolov, N. N. (2004). One-step purification and kinetic properties of the recombinant l-asparaginase from Erwinia carotovora. Biotechnology and Applied Biochemistry, 39, 215–221.

    Article  CAS  Google Scholar 

  47. Labrou, N. E., Papageorgiou, A. C., & Avramis, V. I. (2010). Structure–function relationships and clinical applications of l-asparaginases. Current Medicinal Chemistry, 17, 2183–2195.

    Article  CAS  Google Scholar 

  48. Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40.

    Article  Google Scholar 

  49. Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725–738.

    Article  CAS  Google Scholar 

  50. Roy, A., Yang, J., & Zhang, Y. (2012). COFACTOR: An accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Research, 40, W471–W477.

    Article  CAS  Google Scholar 

  51. Dehouck, Y., Kwasigroch, J. M., Rooman, M., & Gilis, D. (2013). BeAtMuSiC: Prediction of changes in protein–protein binding affinity on mutations. Nucleic Acids Research, 41, W333–W339.

    Article  Google Scholar 

  52. Masso, M., & Vaisman, I. I. (2011) A structure-based computational mutagenesis elucidates the spectrum of stability-activity relationships in proteins. In: Proceedings of 33rd IEEE EMBC (pp. 3225–3228).

  53. Masso, M., & Vaisman, I. I. (2011) Structure-based prediction of protein activity changes: Assessing the impact of single residue replacements. In: Proceedings of 33rd IEEE EMBC (pp. 3221–3224).

  54. Masso, M., & Vaisman, I. I. (2010). AUTO-MUTE: Web-based tools for predicting stability changes in proteins due to single amino acid replacements. Protein Engineering, Design & Selection, 23, 683–687.

    Article  CAS  Google Scholar 

  55. Kotzia, G. A., & Labrou, N. E. (2013). Structural and functional role of Gly281 in l-asparaginase from Erwinia carotovora. Protein & Peptide Letters, 20, 1302–1307.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Roger Hinton, Head of Development & Production, for facilitating these studies, Nigel Allison for advice and critical review of the manuscript, and Trevor Marks, Patrick Kanda, Richard Hesp and Michael Maynard-Smith for helpful discussions. Further thanks go to the entire Development & Production team at PHE Porton.

Author information

Authors and Affiliations

  1. Microbiology Services, Development & Production, Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK

    David Gervais & Nicholas Foote

Authors
  1. David Gervais
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas Foote
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David Gervais.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gervais, D., Foote, N. Recombinant Deamidated Mutants of Erwinia chrysanthemi l-Asparaginase Have Similar or Increased Activity Compared to Wild-Type Enzyme. Mol Biotechnol 56, 865–877 (2014). https://doi.org/10.1007/s12033-014-9766-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-014-9766-9

Keywords

Search

Navigation