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Introducing Intermolecular Interaction to Strengthen the Stability of MnSOD Dimer

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Abstract

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity upon treatments like a wide range of pH, addition of detergent and denaturing agent, exposure to ultraviolet light, and heating up to 50 °C. The enzyme dimer dissociates at 52–55 °C, while its monomer unfolds at 63–67 °C. MnSOD dimeric form is indispensable for the enzyme activity; therefore, strengthening the interactions between the monomers is the most preferred strategy to improve the enzyme stability. However, to date, modification of MnSODSeq at the dimer interface has been unfruitful despite excluding the inner and outer sphere regions that are important to the enzyme activity. Here, a new strategy was developed and K38R-A121E/Y double substitutions were proposed. These mutants displayed similar enzyme activity to the wild type. K38R-A121E dimer was thermally more stable and its monomer stability was similar to the wild type. The thermal stability of K38R-A121Y dimer was similar to the wild type but its monomer was thermally less stable. In addition, the structure of the previously reported L169W mutant was also elucidated. The L169W mutant structure showed that intramolecular modification can decrease flexibility of the MnSODSeq monomer and leads to a less stable enzyme with similar activity to the wild type. Thus, while the enzyme activity depends on arrangement of residues in the dimer interface, the stability appears to depend more on its monomeric architecture. Furthermore, in the L169W structure in complex with azide, which is a specific inhibitor for MnSOD, one of the azide molecules was present in the dimer interface region that previously has been identified to involve in the enzymatic reaction. Nevertheless, the present results show that an MnSODSeq mutant with better thermal stability has been obtained.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Aurora, R., & Rose, G. D. (1998). Helix capping. Protein Science, 7, 21–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Azadmanesh, J., & Borgstahl, G. E. O. (2018). A review of the catalytic mechanism of human manganese superoxide dismutase. Antioxidants, 7, 25–40.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Borgstahl, G., Parge, H., Hickey, M., Beyer, W., Hallewell, R., & Tainer, J. (1992). The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell, 71, 107–118.

    Article  CAS  PubMed  Google Scholar 

  4. Borgstahl, G. E. O., Parge, H. E., Hickey, M. J., Johnson, M. J., Boissinot, M., Hallewell, R. A., Lepock, J. R., Cabelli, D. E., & Tainer, J. A. (1996). Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry, 35, 4287–4297.

    Article  CAS  PubMed  Google Scholar 

  5. Brown, K. A., Didion, S. P., Andresen, J. J., & Faraci, F. M. (2007). Effect of aging, MnSOD deficiency, and genetic background on endothelial function: Evidence for MnSOD haploinsufficiency. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 1941–1946.

    Article  CAS  PubMed  Google Scholar 

  6. Edelheit, O., Hanukoglu, A., & Hanukoglu, I. (2009). Simple and efficient site- directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnology, 8, 1–8.

    Google Scholar 

  7. Edwards, R. A., Whittaker, M. M., Whittaker, J. W., Baker, E. N., & Jameson, G. B. (2001). Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity. Biochemistry, 40, 4622–4632.

    Article  CAS  PubMed  Google Scholar 

  8. Emsley, P., Lohkamp, B., Scott, W., & Cowtan, K. (2010). Features and development of coot. Acta Crystallographica, D 66, 486–501.

    PubMed  Google Scholar 

  9. Ericsson, U., Hallberg, B., Detitta, G., Dekker, N., & Nordlund, P. (2006). Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry, 357, 289–298.

    Article  CAS  PubMed  Google Scholar 

  10. Greenleaf, W. B., Perry, J. J. P., Hearn, A. S., Cabelli, D. E., Lepock, J. R., Stroupe, M. E., Tainer, J. A., Nick, H. S., & Silverman, D. N. (2004). Role of hydrogen bonding in the active site of human manganese superoxide dismutase. Biochemistry, 43, 7038–7045.

    Article  CAS  PubMed  Google Scholar 

  11. Gudiksen, K. L., Gitlin, I., & Whitesides, G. M. (2006). Differentiation of proteins based on characteristic patterns of association and denaturation in solutions of SDS. Proceedings of the National Academy of Sciences, 103, 7968–7972.

    Article  CAS  Google Scholar 

  12. Indrayati, A., Asyarie, S., Suciati, T., & Renoningrum, D. S. (2014) Study on the properties of purified recombinant superoxide dismutase from Staphylococcus equorum, a local isolate from Indonesia. International Journal of Pharmacy and Pharmaceutical Sciences, 6(11), 440-445.

  13. Ismy, J., Sugandi, S., Rachmadi, D., Hardjowijoto, S., & Mustafa, A. (2020). The effect of exogenous superoxide dismutase (SOD) on caspase-3 activation and apoptosis induction in PC-3 prostate cancer cells. Res Rep Urol., 12, 503–508.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Kabsch, W. (2010). Integration, scaling, space-group assignment and post-refinement. Acta Crystallographica, D 66, 133–144.

    PubMed  Google Scholar 

  15. Ludwig, M. L., Metzger, A. L., Pattridge, K. A., & Stallings, W. C. (1991). Manganese superoxide dismutase from Thermus thermophilus: a structural model refined at 1.8 Å resolution. Journal of Molecular Biology, 219, 335–358.

    Article  CAS  PubMed  Google Scholar 

  16. Lee, C.-Y., Liu, Y.-L., Lin, C.-L., Liu, G.-Y., & Hung, H.-C. (2014). Functional roles of the dimer-interface residues in human ornithine decarboxylase. PLoS ONE, 9, e104865–e104875.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Li, C., & Zhou, H.-M. (2011). The role of manganese superoxide dismutase in inflammation defense. Enzyme Research, 2011, 387176.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lyu, P. C., Sherman, J. C., Chen, A., & Kallenbach, N. R. (1991). a-Helix stabilization by natural and unnatural amino acids with alkyl side chains. Proceedings of the National Academy of Sciences, 88, 5317–5320.

    Article  CAS  Google Scholar 

  19. Miller, A.-F. (2012). Superoxide dismutases: Ancient enzymes and new insights. FEBS Letters, 586, 585–595.

    Article  CAS  PubMed  Google Scholar 

  20. Murshudov, G. N., Skuba’k, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., & Vagin, A. A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica, D 67, 355–367.

    PubMed  Google Scholar 

  21. Retnoningrum, D. S., Arumsari, S., Artarini, A., & Ismaya, W. T. (2017). Structure – activity relationship of a recombinant hybrid manganese superoxide dismutase of Staphylococcus saprophyticus/S. equorum. International Journal of Biological Macromolecules, 98, 222–227.

    Article  CAS  PubMed  Google Scholar 

  22. Retnoningrum, D. S., Arumsari, S., Desi, E. S., Tandra, Y. S., Artarini, A., & Ismaya, W. T. (2018). Leu169Trp substitution in MnSOD from Staphylococcus equorum created an active new form of similar resistance to UVC irradiation. Enyzme and Microbial Technology, 118, 13–19.

    Article  CAS  Google Scholar 

  23. Retnoningrum, D. S., Muhammad, A., Fadilah, M. D., Utami, R. A., Artarini, A., & Ismaya, W. T. (2021). Relationship and structural diversity of bacterial manganese superoxide dismutases and the strategy for its application in therapy and cosmetics. Microbiol Indones, 15, 128–134.

    Google Scholar 

  24. Retnoningrum, D. S., Rahayu, A. P., Mulyanti, D., Dita, A., Valerius, O., & Ismaya, W. T. (2016). Unique characteristics of recombinant hybrid manganese superoxide dismutase from Staphylococcus equorum and S. saprophyticus. Protein Journal, 35, 136–144.

    Article  CAS  PubMed  Google Scholar 

  25. Retnoningrum, D. S., Yoshida, H., Arumsari, S., Kamitori, S., & Ismaya, W. T. (2018). The first crystal structure of manganese superoxide dismutase from the genus Staphylococcus. Acta Crystallographica Section F, 74, 135–142.

    CAS  Google Scholar 

  26. Retnoningrum, D. S., Yoshida, H., Razani, M., Meidianto, V. F., Hartanto, A., Artarini, A., & Ismaya, W. T. (2021). Unprecedented role of the N73–F124 pair in the Staphylococcus equorum MnSOD Activity. Current Enzyme Inhibition, 17, 2–8.

    Article  CAS  Google Scholar 

  27. Retnoningrum, D. S., Yoshida, H., Razani, M. D., Muliadi, R., Meidianto, V. F., Artarini, A., & Ismaya, W. T. (2021). The role of S126 in the Staphylococcus equorum MnSOD activity and stability. Journal of Structural Biology, 213, 107731–107734.

    Article  CAS  PubMed  Google Scholar 

  28. S˘ali, D., Bycroft, M., & Fersht, A. R. (1988). Stabilization of protein structure by interaction of α-helix dipole with a charged side chain. Nature, 335, 740–743.

    Article  PubMed  Google Scholar 

  29. Ściskalska, M., Ołdakowska, M., Marek, G., & Milnerowicz, H. (2020). Changes in the activity and concentration of superoxide dismutase isoenzymes (Cu/Zn SOD, MnSOD) in the blood of healthy subjects and patients with acute pancreatitis. Antioxidants, 9, 948–963.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Sharma, S., Bhattarai, S., Ara, H., Sun, G., Clair, D. K. S., Bhuiyan, M. S., Kevil, C., Watts, M. N., Dominic, P., Shimizu, T., McCarthy, K. J., Sun, H., Panchatcharam, M., & Miriyala, S. (2020). SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy. Redox Biology, 37, 101740–101753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sheng, Y., Abreu, I. A., Cabelli, D. E., Maroney, M. J., Miller, A.-F., Teixeira, M., & Valentine, J. S. (2014). Superoxide dismutases and superoxide reductases. Chemical Reviews, 114, 3854–3918.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sheng, Y., Durazo, A., Schumacher, M., Gralla, E. B., Cascio, D., Cabelli, D. E., & Valentine, J. S. (2013). Tetramerization reinforces the dimer interface of MnSOD. PLoS ONE, 8, 62446. https://doi.org/10.61371/journal.pone.0062446

    Article  Google Scholar 

  33. Sivaramakrishnan, S., Spink, B. J., Sim, A. Y. L., Doniach, S., & Spudich, J. A. (2008). Dynamic charge interactions create surprising rigidity in the ER/K α-helical protein motif. Proceedings of the National Academy of Sciences, 105, 13356–13361.

    Article  CAS  Google Scholar 

  34. Vagin, A., & Teplyakov, A. (2010). Molecular replacement with MOLREP. Acta Crystallographica, D 66, 22–25.

    PubMed  Google Scholar 

  35. Whittaker, M. M., & Whittaker, J. W. (1998). A glutamate bridge is essential for dimer stability and metal selectivity in manganese superoxide dismutase. Journal of Biological Chemistry, 273, 22188–22193.

    Article  CAS  PubMed  Google Scholar 

  36. Winn, M., Ballard, C., Cowtan, K., Dodson, E., Emsley, P., Evans, P., Keegan, R., Krissinel, E., Leslie, A., McCoy, A., McNicholas, S., Murshudov, G., Pannu, N., Potterton, E., Powell, H., Read, R., Vagin, A., & Wilson, K. (2011). Overview of the CCP4 suite and current developments. Acta Crystallographica, D 67, 235–242.

    PubMed  Google Scholar 

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Funding

The work has been supported by The World Class Research program, Ministry of Research, Technology and Higher Education (2020), by Dexa Medica, and by Department of Basic Life Science, Faculty of Medicine, Kagawa University. We thank the staffs of the Photon Factory (KEK, Tsukuba, Japan) for the support of data collection.

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Authors and Affiliations

  1. Laboratory of Pharmaceutical Biotechnology, Pharmaceutics Research Group, School of Pharmacy, Institut Teknologi Bandung, Ganesha 10, Bandung, 40132, West Java, Indonesia

    Debbie S. Retnoningrum, Ismiana Pajatiwi, Rahmat Muliadi, Ratna A. Utami & Anita Artarini

  2. Department of Basic Life Science, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa, 761-0793, Japan

    Hiromi Yoshida

  3. Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Industri Selatan V Blok PP-7, Cikarang, 17750, West Java, Indonesia

    Wangsa T. Ismaya

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  1. Debbie S. Retnoningrum
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Contributions

DSR: conceptualization, funding acquisition; HY: methodology, formal analysis and investigation, manuscript review and editing; IP: formal analysis and investigation, original draft preparation; RM: formal analysis and investigation, original draft preparation; RAU: methodology, manuscript review and editing, supervision; AA: methodology, manuscript review and editing, funding acquisition, supervision; WTI: methodology, original draft preparation, manuscript review and editing, supervision.

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Correspondence to Anita Artarini.

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Debbie S. Retnoningrum deceased during completion of the works.

This manuscript is dedicated to D. Retnoningrum, who had initiated the works on MnSOD from S. equorum in Indonesia.

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Retnoningrum, D.S., Yoshida, H., Pajatiwi, I. et al. Introducing Intermolecular Interaction to Strengthen the Stability of MnSOD Dimer. Appl Biochem Biotechnol 195, 4537–4551 (2023). https://doi.org/10.1007/s12010-023-04347-7

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