Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

  • Lee, Jae Taek (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Lee, Seung Sik (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Mondal, Suvendu (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Tripathi, Bhumi Nath (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Kim, Siu (Division of Applied Life Science (Brain Korea 21 Program), Gyeongsang National University) ;
  • Lee, Keun Woo (Division of Applied Life Science (Brain Korea 21 Program), Gyeongsang National University) ;
  • Hong, Sung Hyun (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Bai, Hyoung-Woo (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Cho, Jae-Young (Department of Bioenvironmental Chemistry, Chonbuk National University) ;
  • Chung, Byung Yeoup (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI))
  • Received : 2016.02.18
  • Accepted : 2016.06.22
  • Published : 2016.08.31
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Abstract

Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of $Ser^{78}$ to $Cys^{78}$ resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of $Cys^{78}$ in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced1 survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone.

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References

  1. An, B.C., Lee, S.S., Lee, E.M., Lee, J.T., Wi, S.G., Jung, H.S., Park, W., and Chung, B.Y. (2010). A new antioxidant with dual functions as a peroxidase and chaperone in Pseudomonas aeruginosa. Mol. Cells 29, 145-151. https://doi.org/10.1007/s10059-010-0023-1
  2. An, B.C., Lee, S.S., Lee, E.M., Lee, J.T., Wi, S.G., Jung, H.S., Park, W., Lee, S.Y., and Chung, B.Y. (2011). Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress. Cell Stress Chap. 16, 317-328. https://doi.org/10.1007/s12192-010-0243-5
  3. An, B.C., Lee, S.S., Jung, H.S., Kim, J.Y., Lee, Y., Lee, K.W., Lee, S.Y., Tripathi, B. N., and Chung, B.Y. (2015). An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone. FEBS Lett. 589, 2831-2840. https://doi.org/10.1016/j.febslet.2015.07.046
  4. Angelucci, F., Saccoccia, F., Ardini, M., Boumis, G., Brunori, M., Di Leandro, L., Ippoliti, R., Miele, A.E., Natoli, G., Scotti, S., et al. (2013). Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis. J. Mol. Biol. 425, 4556-4568. https://doi.org/10.1016/j.jmb.2013.09.002
  5. Bhatt, I., and Tripathi, B.N. (2011). Plant peroxiredoxin: catalytic mechanisms, functional significance and future perspectives. Biotechnol. Adv. 29, 850-859. https://doi.org/10.1016/j.biotechadv.2011.07.002
  6. Bryk, R., Lima, C.D., Erdjument-Bromage, H., Tempst, P., and Nathan, C. (2002). Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein. Science 295, 1073-1077. https://doi.org/10.1126/science.1067798
  7. Chuang, M.H., Wu, M.S., Lo, W.L., Lin, J.T., Wong, C.H., and Chiou, S.H. (2006). The antioxidant protein alkyl hydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function. Proc. Natl. Acad. Sci. USA 103, 2552-2557. https://doi.org/10.1073/pnas.0510770103
  8. Gnanasekar, M., Dakshinamoorthy, G., and Ramaswamy, K. (2009). Translationally controlled tumor protein is a novel heat shock protein with chaperone-like activity. Biochem. Biophy. Res. Comm. 386, 333-337. https://doi.org/10.1016/j.bbrc.2009.06.028
  9. Hall, A., Karplus, P.A., and Poole, L.B. (2009). Typical 2-Cys peroxiredions-structures, mechanisms and functions. FEBS J. 276, 2469-2477. https://doi.org/10.1111/j.1742-4658.2009.06985.x
  10. Huang, C.H., Chuang, M.H., Wu, Y.H., Chuang, W.C., Jhuang, P.J., and Chiou, S.H. (2010). Characterization of site-specific mutants of alkylhydroperxide reductase with dual functionality from Helicobacter pylori. J. Biochem. 147, 661-669. https://doi.org/10.1093/jb/mvp209
  11. Ito, H., Kamei, K., Iwamoto, I., Inaguma, Y., Nohara, D., and Kato, K. (2001). Phosphorylation-induced change of the oligomerization state of alpha B-crystallin. J. Biol. Chem. 276, 5346-5352. https://doi.org/10.1074/jbc.M009004200
  12. Jang, H.H., Lee, K.O., Chi, Y.H., Jung, B.G., Park, S.K., Park, J.H., Lee, J.R., Lee, S.S., Moon, J.C., Yun, J.W., et al. (2004). Two enzymes in one, two yeast peroxiredoxins display oxidative stressdependent switching from a peroxidase to a molecular chaperone function. Cell 117, 625-635. https://doi.org/10.1016/j.cell.2004.05.002
  13. Konig, J., Galliardt, H., Jutte, P., Schaper, S., Dittmann, L., and Dietz, K.J. (2013). The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone. J. Exp. Bot. 64, 3483-3497. https://doi.org/10.1093/jxb/ert184
  14. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291. https://doi.org/10.1107/S0021889892009944
  15. Lee, W., Choi, K.S., Riddell, J., Ip, C., Ghosh, D., Park, J.H., and Park, Y.M. (2007). Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2. J. Biol. Chem. 282, 22011-22022. https://doi.org/10.1074/jbc.M610330200
  16. Lee, E.M., Lee, S.S., Tripathi, B.N., Jung, H.S., Cao, G.P., Lee, Y., Singh, S., Hong, S.H., Lee, K.W., Lee, S.Y., et al. (2015). Sitedirected mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions. Ann. Bot.116, 713-725. https://doi.org/10.1093/aob/mcv094
  17. Mayer, M.P., and Bukau, B. (2005). Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670-684. https://doi.org/10.1007/s00018-004-4464-6
  18. Moon, J.C., Hah, Y.S., Kim, W.Y., Jung, B.G., Jang, H.H., Lee, J.R., Kim, S.Y., Lee, Y.M., Jeon, M.K., Kim, C.W., et al. (2005). Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin isotype II that enhances HeLa cell resistance to $H_2O_2$-induced cell death. J. Biol. Chem. 280, 28775-28784. https://doi.org/10.1074/jbc.M505362200
  19. Nelson, K.J., and Parsonage, D. (2011). Measurement of peroxiredoxin activity. Curr. Protoc. Toxicol. 49, 7.10.1-7.10.28. https://doi.org/10.1002/0471140856.tx0710s49
  20. Ochsner, U.A., Vasil, M.L., Alsabbagh, E., Parvatiyar, K., and Hassett, D. (2000). Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyRdependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182, 4533-4544. https://doi.org/10.1128/JB.182.16.4533-4544.2000
  21. Park, J.W., Piszczek, G., Rhee, S.G., and Chock, P.B. (2011). Glutathionylation of peroxiredoxin induces decamer to dimers dissociation with concomitant loss of chaperone activity. Biochemistry 50, 3204-3210. https://doi.org/10.1021/bi101373h
  22. Parsonage, D., Youngblood, D.S., Ganapathy, N.S., Wood, Z.A., Karpus, A.P., and Poole, L.B. (2005). Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin. Biochemistry 44, 10583-10592. https://doi.org/10.1021/bi050448i
  23. Parsonage, D., Karplus, P.A., and Poole, L.B. (2008). Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin. Proc. Natl. Acad. Sci. USA 105, 8209-8214. https://doi.org/10.1073/pnas.0708308105
  24. Poole, L.B. (1996). Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium 2. Cystine disulfides involved in catalysis of peroxide reduction. Biochemistry 35, 65-75. https://doi.org/10.1021/bi951888k
  25. Saccoccia, F., Di Micco, P., Boumis, G., Brunori, M., Koutris, I., Miele, A.E., Morea, V., Sriratana, P., Williams, D.L., Bellelli, A., et al. (2012). Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin. Structure 20, 429-439. https://doi.org/10.1016/j.str.2012.01.004
  26. Sharma, K.K., Kaur, H., Kumar, G.S., and Kester, K. (1998). Interaction of 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid with alpha-crystallin. J. Biol. Chem. 273, 8965-8970. https://doi.org/10.1074/jbc.273.15.8965
  27. Tairum, C.A., de Oliveira, M.A., Horta, B.B., Zara, F.J., and Netto, L.E.S. (2012). Disulfide biochemistry in 2-Cys peroxiredoxin: requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin. J. Mol. Biol. 424, 28-41. https://doi.org/10.1016/j.jmb.2012.09.008
  28. Tripathi, B.N., Bhatt, I., and Dietz, K.J. (2009). Peroxiredoxins: a less studied component of hydrogen peroxide detoxification in photosynthetic organisms. Protoplasma 235, 3-15. https://doi.org/10.1007/s00709-009-0032-0
  29. Wiederstein, M., and Sippl, M.J. (2007). ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 35, 407-410.
  30. Woo, M.-H., Kim, M.S., Chung, N., and Kim, J.-S. (2014). Expression and characterization of a novel 2-deoxyribose-5-phosphate aldolase from Haemophilus influenzae Rd KW20. J. Korean Soc. Appl. Biol. Chem. 57, 655-660. https://doi.org/10.1007/s13765-014-4231-9
  31. Wood, Z.A., Poole, L.B., Hantgan, R.R., and Karpus, A.P. (2002). Dimers to doughnut: redox sensitive oligomerization of 2-cysteine peroxiredoxins. Biochemistry 41, 5493-5505. https://doi.org/10.1021/bi012173m
  32. Wood, Z.A., Schroder, E., Robin, H.J., and Poole, L.B. (2003). Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 32-40. https://doi.org/10.1016/S0968-0004(02)00003-8

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