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:: Volume 1, Issue 2 (8-2025) ::
Journalaer 2025, 1(2): 23-36 Back to browse issues page
Rare codons analysis, the structure determination, and investigation of the active site of L-asparaginase II of B. megaterium
Safa Lotfi * , Mojtaba Mortazavi
Abstract:   (339 Views)
Introduction: Currently, L-asparaginase II of Escherichia coli and Erwinia chrysanthemi are prescribed for the treatment of acute lymphoblastic leukemia (ALL) in children. However, due to their short half-life and side effects related to immunogenicity and glutaminase activity, the research to find new bacterial sources of L-asparaginase with higher therapeutic effects and minimal side effects has continued. In this regard, the L-asparaginase II of Bacillus megaterium has been studied in this research.
Materials & methods: Rare codons of the L-asparaginase II gene of B. megaterium were identified with ATGme server and amino acid residues related to these codons were determined. The third protein structure was predicted using three homology modeling-based servers and the quality of the models was studied with ERRAT and Verify3D servers. The amino acid residues of the active site were determined with PyMol software.
Results: The results showed that this gene has 73 rare codons and 27 very rare codons. All three servers (SWISS-MODEL, Phyre2, and I-TASSER) used L-asparaginase II of E. chrysanthemi as the template, and the model created by SWISS-MODEL had higher accuracy and quality than the other two models. The comparison of the active site residues of this enzyme with L-asparaginase II of E. chrysanthemi indicated that out of a total of 8 residues, 5 amino acids are similar and 3 amino acids are different. Glu63, Thr95 and Ala120 are replaced with Gln103, Ser135, and Ser160 in L-asparaginase II of B. megaterium. According to the previous published reports, the presence of glutamine instead of glutamate in the L-asparaginase II active site reduces glutaminase activity. The analysis of amino acids with rare codons showed that all three serine of active site possess rare codons. Therefore, due to the high number of rare codons and the fact that some of them are related to the active site residues, during the process of codon optimization, a detailed and comprehensive study should be carried out to ensure high protein production and its solubility in the E. coli expression system.

Conclusion: Summing up, the obtained results can help the process of designing and producing the L-asparaginase II enzyme with higher therapeutic properties and lower side effects.
Keywords: Acute lymphoblastic leukemia (ALL), L-asparaginase II, Bacillus megaterium, Rare codons, SWISS-MODEL
Full-Text [PDF 1438 kb]   (111 Downloads)    
Type of Study: Research | Subject: Special
Received: 2026/02/24 | Accepted: 2025/08/1 | Published: 2025/08/1
References
1. 1. Chand, S., Mahajan, R.V., Prasad, J.P., Sahoo, D.K., Mihooliya, K.N., Dhar, M.S. and Sharma, G., 2020. A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization & industrial applications. Biotechnology and Applied Biochemistry. 67(4): 619-647. doi: 10.1002/bab.1888
2. 2. Schrappe, M., Reiter, A., Ludwig, W.D., Harbott, J., Zimmermann, M., Hiddemann, W., Niemeyer, C., Henze, G., Feldges, A., Zintl, F., Kornhuber, B., Ritter, J., Welte, K., Gadner, H. and Riehm, H., 2000. Improved outcome in childhood acute lymphoblastic leukemia despite reduced use of anthracyclines and cranial radiotherapy: results of trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM Study Group. Blood. 95(11): 3310-3322.
3. 3. Kiriyama, Y., Kubota, M., Takimoto, T., Kitoh, T., Tanizawa, A., Akiyama, Y. and Mikawa, H., 1989. Biochemical characterization of U937 cells resistant to
4. L-asparaginase: the role of asparagine synthetase. Leukemia. 3(4): 294-297.
5. 4. Stams, W.A., den Boer, M.L., Beverloo, H.B., Meijerink, J.P., Stigter, R.L., van Wering, E.R., Janka Schaub, G.E., Slater, R. and Pieters, R., 2003. Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21)+ pediatric ALL. Blood. 101(7): 2743-2747. doi: 10.1182/ blood-2002-08-2446
6. 5. Cachumba, J.J., Antunes, F.A., Peres, G.F., Brumano, L.P., Santos, J.C. and Da Silva, S.S., 2016. Current applications and different approaches for microbial
7. l-asparaginase production. Brazilian journal of microbiology. 47(1): 77-85. doi: 10.1016/j.bjm.2016.10. 004
8. 6. Mortazavi, M., Torkzadeh-Mahani, M., Kargar, F., Nezafat, N. and Ghasemi, Y., 2020. In silico analysis of codon usage and rare codon clusters in the halophilic bacteria L-asparaginase. Biologia. 75(1): 151-160. https://doi.org/10.2478/s11756-019-00324-w
9. 7. Shrivastava, A., Khan, A.A., Khurshid, M., Kalam, M.A., Jain, S.K. and Singhal, P.K., 2016. Recent developments in L-asparaginase discovery and its potential as anticancer agent. Critical Reviews in Oncology/Hematology. 100: 1-10. doi: 10.1016/j.crit revonc.2015.01.002
10. 8. Brumano, L.P., da Silva, F.V.S., Costa-Silva, T.A., Apolinario, A.C., Santos, J., Kleingesinds, E.K., Monteiro, G., Rangel-Yagui, C.O., Benyahia, B. and Junior, A.P., 2018. Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles. Frontiers in Bioengineering and Biotechnology. 6: 212.doi: 10.3389/fbioe.2018.00212
11. 9. Verma, N., Kumar, K., Kaur, G. and Anand, S., 2007. L-asparaginase: a promising chemotherapeutic agent. Critical Reviews in Biotechnology. 27(1): 45-62. doi: 10. 1080/07388550601173926
12. 10. Liu, Y., 2020. A code within the genetic code: codon usage regulates co-translational protein folding. Cell Communication and Signaling. 18(1): 145. doi: 10.1186/ s12964-020-00642-6
13. 11. Wang, Y., Li, C., Khan, M.R., Wang, Y., Ruan, Y., Zhao, B., Zhang, B., Ma, X., Zhang, K., Zhao, X., Ye, G., Guo, X., Feng, G., He, L. and Ma, G., 2016. An Engineered Rare Codon Device for Optimization of Metabolic Pathways. Scientific Reports. 6: 20608. doi: 10.1038/ srep20608
14. 12. Henry, I. and Sharp, P.M., 2007. Predicting gene expression level from codon usage bias. Molecular Biology and Evolution. 24(1): 10-12. doi: 10.1093/mol bev/msl148
15. 13. Rosano, G.L. and Ceccarelli, E.A., 2009. Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial Cell Factories. 8: 41. doi: 10.1186/1475-2859-8-41
16. 14. Takenaka, Y., Haga, N., Harumoto, T., Matsuura, T. and Mitsui, Y., 2002. Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene. Gene. 284(1-2): 233-240. doi: 10.1016/s0378-1119(01)00886-1
17. 15. Kane, J.F., 1995. Effects of rare codon clusters on high level expression of heterologous proteins in Escherichia coli. Current Opinion in Biotechnology. 6(5): 494-500. doi: 10.1016/0958-1669(95)80082-4
18. 16. Kim, S. and Lee, S.B., 2006. Rare codon clusters at
19. 5'-end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expression and Purification. 50(1): 49-57. doi: 10.1016/j.pep.2006.07.014
20. 17. Correddu, D., Montano Lopez, J.J., Angermayr, S.A., Middleditch, M.J., Payne, L.S. and Leung, I.K.H., 2020. Effect of consecutive rare codons on the recombinant production of human proteins in Escherichia coli. IUBMB Life. 72(2): 266-274. doi: 10.1002/iub.2162
21. 18. Deane, C.M. and Saunders, R., 2011. The imprint of codons on protein structure. The Biotechnology Journal. 6(6): 641-649. doi: 10.1002/biot.201000329
22. 19. Zhang, G., Hubalewska, M. and Ignatova, Z., 2009. Transient ribosomal attenuation coordinates protein synthesis and co-translational folding. Nature Structural & Molecular Biology. 16(3): 274-280. doi: 10. 1038/nsmb.1554
23. 20. Liu, L., Yang, H., Shin, H.D., Chen, R.R., Li, J., Du, G. and Chen, J., 2013. How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioengineered. 4(4): 212-223. doi: 10.4161 /bioe.24761
24. 21. Roth, A., Anisimova, M. and Cannarozzi, G.M., 2012. Measuring codon usage bias. In: Cannarozzi GM, Schneider A, editors. Codon Evolution: Mechanisms and Models: Oxford University Press.
25. 22. Parameswaran, B., Papamichael, E., Varjani, S. and Raveendran, S., 2019. Introduction to Green Bioprocesses: Industrial Enzymes for Food Applications. In: Parameswaran, B., Varjani, S. and Raveendran, S., (eds) Green Bio-processes. Energy, Environment, and Sustainability. Singapore: Springer Singapore.
26. 23. Ghasemian, A., Al-Marzoqi, A.H., Al-Abodi, H.R., Alghanimi, Y.K., Kadhum, S.A., Shokouhi Mostafavi, S.K. and Fattahi, A., 2019. Bacterial l-asparaginases for cancer therapy: Current knowledge and future perspectives. Journal of Cellular Physiology. 234(11): 19271-19279. doi: 10.1002/jcp.28563
27. 24. Li, X., Zhang, X., Xu, S., Xu, M., Yang, T., Wang, L., Zhang, H., Fang, H., Osire, T. and Rao, Z., 2019. Insight into the thermostability of thermophilic
28. L-asparaginase and non-thermophilic L-asparaginase II through bioinformatics and structural analysis. Applied Microbiology and Biotechnology. 103(17): 7055-7070. doi: 10.1007/s00253-019-09967-w
29. 25. da Cunha, M.C., Dos Santos Aguilar, J.G., de Melo, R.R., Nagamatsu, S.T., Ali, F., de Castro, R.J.S. and Sato, H.H., 2019. Fungal L-asparaginase: Strategies for production and food applications. Food Research International. 126: 108658. doi: 10.1016/j.foodres.2019. 108658
30. 26. Nakamura, Y., Gojobori, T. and Ikemura, T., 2000. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research. 28(1): 292. doi: 10.1093/nar/28.1.292
31. 27. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. and Schwede, T., 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Research 46(W1): W296-W303. doi: 10.1093/nar/gky427
32. 28. Bienert, S., Waterhouse, A., de Beer, T.A., Tauriello, G., Studer, G., Bordoli, L. and Schwede, T., 2017. The SWISS-MODEL Repository-new features and functionality. Nucleic Acids Research. 45(D1): D313-D319. doi: 10. 1093/nar/gkw1132
33. 29. Schwede, T., Kopp, J., Guex, N. and Peitsch, M.C., 2003. SWISS-MODEL: An automated protein homology modeling server. Nucleic Acids Research. 31(13): 3381-3385. doi: 10.1093/nar/gkg520
34. 30. Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J., 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols. 10(6): 845-858. doi: 10.1038/nprot.2015.053
35. 31. Roy, A., Kucukural, A. and Zhang, Y., 2010.
36. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols. 5(4): 725-738. doi: 10.1038/nprot.2010.5
37. 32. Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J. and Zhang, Y., 2015. The I-TASSER Suite: protein structure and function prediction. Nature methods. 12(1): 7-8. doi: 10.1038/nmeth.3213
38. 33. Wu, S. and Zhang, Y., 2007. LOMETS: a local meta threading-server for protein structure prediction. Nucleic acids research. 35(10): 3375-3382. doi: 10.1093/nar/gk m251
39. 34. Colovos, C. and Yeates, T.O., 1993. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Science. 2(9): 1511-1519. doi: 10. 1002/pro.5560020916
40. 35. Luthy, R., Bowie, J.U. and Eisenberg, D., 1992. Assessment of protein models with three-dimensional profiles. Nature. 356(6364): 83-85. doi: 10.1038/3560 83a0
41. 36. Lubkowski, J., Dauter, M., Aghaiypour, K., Wlodawer, A. and Dauter, Z., 2003. Atomic resolution structure of Erwinia chrysanthemi L-asparaginase. Acta Crystallogr D Biology Crystallogr. 59(Pt 1): 84-92. doi: 10.1107/s0907444902019443
42. 37. Nguyen, H.A., Su, Y. and Lavie, A., 2016. Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity. Journal of Biological Chemistry 291(34): 17664-17676. doi: 10.1074/jbc.M116.728485
43. 38. Papageorgiou, A.C., Posypanova, G.A., Andersson, C.S., Sokolov, N.N. and Krasotkina, J., 2008. Structural and functional insights into Erwinia carotovora
44. L-asparaginase. FEBS Journal. 275(17): 4306-4316. doi: 10.1111/j.1742-4658.2008.06574.x
45. 39. Jaskolski, M., Kozak, M., Lubkowski, J., Palm, G. and Wlodawer, A., 2001. Structures of two highly homologous bacterial L-asparaginases: a case of enantiomorphic space groups. Acta Crystallogr D Biology Crystallogr. 57(Pt 3): 369-377. doi: 10.1107/s090744490 0020175
46. 40. Kravchenko, O.V., Kislitsin, Y.A., Popov, A.N., Nikonov, S.V. and Kuranova, I.P., 2008. Three dimensional structures of L-asparaginase from Erwinia carotovora complexed with aspartate and glutamate Acta Crystallogr D Biology Crystallogr. 64(Pt 3): 248-256. doi: 10.1107/S0907444907065766
47. 41. Van Trimpont, M., Schalk, A.M., De Visser, Y., Nguyen, H.A., Reunes, L., Vandemeulebroecke, K., Peeters, E., Su, Y., Lee, H., Lorenzi, P.L., Chan, W.K., Mondelaers, V., De Moerloose, B., Lammens, T., Goossens, S., Van Vlierberghe, P. and Lavie, A., 2023. In vivo stabilization of a less toxic asparaginase variant leads to a durable antitumor response in acute leukemia. Haematologica. 108(2): 409-419. doi: 10.3324/haematol. 2022.281390
48. 42. Koshafar, A., Savari, A., Sakhaei, N., Archangi, B. and Karimi Organi, F., 2019. Evaluation of carcinogenicity and non-carcinogenicity of heavy metals in the dominant muscle of Bahmanshir River. Journal of Animal Environment. 11(4): 155-162. (In Persian)
49. 43. Hassanpour, S.H. and Dehghani, M., 2017. Review of cancer from perspective of molecular. Journal of Cancer Research and Practice. 4(4): 127-129. https://doi.org/10. 1016/j.jcrpr.2017.07.001
50. 44. Zohrevand, A., Sadeghi, M.S., Emtyazjoo, M. and Hejazi, H., 2023. Anticancer effects of coral extract of Sinularia compressa on human gastric adenocarcinoma cell line (AGS). Journal of Animal Environment. 15(1): 273-278. doi: 10.22034/AEJ.2022.338193.2789 (In Persian)
51. 45. Debela, D.T., Muzazu, S.G., Heraro, K.D., Ndalama, M.T., Mesele, B.W., Haile, D.C., Kitui, S.K. and Manyazewal, T., 2021. New approaches and procedures for cancer treatment: Current perspectives. Sage Open Medicine. 9: 20503121211034366. doi: 10.1177/205031 21211034366
52. 46. Safary, A., Moniri, R., Hamzeh-Mivehroud, M. and Dastmalchi, S., 2019. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. Bioimpacts. 9(1): 15-23. doi: 10.15171/bi.2019.03
53. 47. Jia, M., Xu, M., He, B. and Rao, Z., 2013. Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06. Journal of Agricultural and Food Chemistry. 61(39): 9428-9434. doi: 10.1021/jf402636w
54. 48. de Araújo, T.S., Scapin, S.M.N., de Andrade, W., Fasciotti, M., de Magalhães, M.T.Q., Almeida, M.S. and Lima, L.M.T.R., 2021. Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2. Biophysical Chemistry. 271: 106554. doi: 10.1016/j.bpc. 2021.106554
55. 49. Duval, M., Suciu, S., Ferster, A., Rialland, X., Nelken, B., Lutz, P., Benoit, Y., Robert, A., Manel, A.M., Vilmer, E., Otten, J. and Philippe, N., 2002. Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer-Children's Leukemia Group phase 3 trial. Blood. 99(8): 2734-2739. doi: 10.1182/blood.v99.8.2734
56. 50. Sudhir, A.P., Dave, B.R., Prajapati, A.S., Panchal, K., Patel, D. and Subramanian, R.B., 2014. Characterization of a recombinant glutaminase-free
57. L-asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis. Applied Biochemistry and Biotechnology. 174(7): 2504-2515. doi: 10.1007/s12010-014-1200-z
58. 51. Pokrovskaya, M.V., Aleksandrova, S.S., Pokrovsky, V.S., Veselovsky, A.V., Grishin, D.V., Abakumova, O.Y., Podobed, O.V., Mishin, A.A., Zhdanov, D.D. and Sokolov, N.N., 2015. Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site directed mutagenesis. Molecular Biotechnology. 57(3): 251-264. doi: 10.1007/s12033-014-9819-0
59. 52. Lubkowski, J. and Wlodawer, A., 2021. Structural and biochemical properties of L-asparaginase. FEBS Journal. 288(14): 4183-4209. doi: 10.1111/febs.16042
60. 53. Aghaiypour, K., Wlodawer, A. and Lubkowski, J., 2001. Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. Biochemistry. 40(19): 5655-5664. doi: 10.1021/bi0029 595
61. 54. Kumar, D. and Sobha, K., 2012. L-Asparaginase from Microbes: a Comprehensive Review. Advances in Bioresearch. 3: 137-157.
62. 55. Derst, C., Henseling, J. and Rohm, K.H., 2000. Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein Science. 9(10): 2009-2017. doi: 10.1110/ps.9.10. 2009
63. 56. Swain, A.L., Jaskolski, M., Housset, D., Rao, J.K. and Wlodawer, A., 1993. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proceedings of the National Academy of Sciences of the United States of America. 90(4): 1474-1478. doi: 10. 1073/pnas.90.4.1474
64. 57. Tripathy, R.K., Anakha, J. and Pande, A.H., 2023. Towards development of biobetter: L-asparaginase a case study. Biochimica et Biophysica Acta- General Subjects. 1868(1): 130499. doi: 10.1016/j.bbagen.2023. 130499
65. 58. Chan, W.K., Lorenzi, P.L., Anishkin, A., Purwaha, P., Rogers, D.M., Sukharev, S., Rempe, S.B. and Weinstein, J.N., 2014. The glutaminase activity of
66. L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood. 123(23): 3596-3606. doi: 10.1182/blood-2013-10-535112
67. 59. Ardalan, N., Mirzaie, S., Sepahi, A.A. and Khavari Nejad, R.A., 2018. Novel mutant of Escherichia coli asparaginase II to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and QM-MM studies. Medical Hypotheses. 112: 7-17. doi: 10.1016/j.mehy. 2018.01.004
68. 60. Mauro, V.P., 2018. Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations. BioDrugs. 32(1): 69-81. doi: 10.1007/s40259-018-0261-x
69. 61. Parvathy, S.T., Udayasuriyan, V. and Bhadana, V., 2022. Codon usage bias. Molecular Biology Reports. 49(1): 539-565. doi: 10.1007/s11033-021-06749-4
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Lotfi S, Mortazavi M. Rare codons analysis, the structure determination, and investigation of the active site of L-asparaginase II of B. megaterium. Journalaer 2025; 1 (2) :23-36
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Volume 1, Issue 2 (8-2025) Back to browse issues page
پژوهش های محیط زیست جانوری Journal of Animal Environmental Research
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