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American Journal of Medical and Biological Research. 2016, 4(3), 42-52
DOI: 10.12691/AJMBR-4-3-2
Original Research

Role of Some Metal Ions on Steady–state Kinetics of Engineered Wild–type and Manganese (II) Binding Site Mutants of Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3)

Usenobong F. Ufot1, , Aniefiok E. Ite2, 3, Idorenyin H. Usoh1 and Monday I. Akpanabiatu4

1Department of Biological Sciences, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

2Department of Chemistry, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

3Research and Development Unit, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

4Department of Biochemistry, University of Uyo, P. M. B. 1017, Uyo, Akwa Ibom State, Nigeria

Pub. Date: August 06, 2016
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Usenobong F. Ufot, Aniefiok E. Ite, Idorenyin H. Usoh and Monday I. Akpanabiatu. Role of Some Metal Ions on Steady–state Kinetics of Engineered Wild–type and Manganese (II) Binding Site Mutants of Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3). American Journal of Medical and Biological Research. 2016; 4(3):42-52. doi: 10.12691/AJMBR-4-3-2

Abstract

This study investigated the steady-state kinetics of engineered wild-type and manganese (II) binding site mutants of recombinant Phlebia radiata manganese peroxidase 3(rPr-MnP3). The effect (activation or inhibition) of some metal ions (Co2+, Zn2+ Cu2+ and Na+) on the activity of rPr-MnP3 enzymes was also studied. The results obtained showed that the rPr-MnP3 mutants in which the metal binding functionality has been largely lost have been created. Na+ (mono-valent ion) and Co2+showed similar characteristics by exhibiting stimulatory effects on the activity of wild-type rPr-MnP3. However, Cu2+ and Zn2+ had mixed inhibitory effects on wild-type and mutants (E40H, E44H, E40H/E44H). It was observed that Cu2+ was by far the strongest inhibitor of engineered rPr-MnP3 enzymes while Co2+ exhibited a non-competitive inhibitory effect on the double mutant (E40H/E44H) and D186H activities. In addition, Zn2+ and Cu2+also had non-competitive inhibitory effect on D186H mutant enzyme activity. The results obtained further showed that the competitive inhibitory effect of Cu2+observed in other rPr-MnP3 enzymes is largely removed in D186H mutant enzyme. Generally, histidine substitution retained a strong selectivity for Cu2+ as competitive inhibitor. Zn2+ being generally non-competitive suggest involvement of sites other than the Mn (II) binding site. This study showed that rPr-MnP3 enzymes function with alternate ligands in the Mn2+ binding site and does not have absolute obligate requirement for all carboxylate ligand set.

Keywords

peroxidase, Phlebiaradiata, steady-state, wild-type, mutants, metal ions, inhibitors

Copyright

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References

[1]  Hatakka, A. I., and A. K. Uusi-Rauva, “Degradation of 14C-labelled poplar wood lignin by selected white-rot fungi,” European journal of applied microbiology and biotechnology, 17 (4). 235-242, 1983.
 
[2]  Lundell, T., A. Leonowicz, J. Rogalski, and A. Hatakka, “Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid,” Applied and Environmental Microbiology, 56 (9). 2623-2629, 1990.
 
[3]  Vares, T., M. Kalsi, and A. Hatakka, “Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw,” Applied and Environmental Microbiology, 61 (10). 3515-3520, 1995.
 
[4]  Hatakka, A., T. Lundell, M. Hofrichter, and P. Maijala, “Manganese Peroxidase and Its Role in the Degradation of Wood Lignin,” Applications of Enzymes to Lignocellulosics, ACS Symposium Series 855, S. D. Mansfield and J. N. Saddler, eds., pp. 230-243: American Chemical Society, 2003.
 
[5]  Niemenmaa, O., A. Uusi-Rauva, and A. Hatakka, “Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata,” Archives of Microbiology, 185 (4). 307-315, 2006.
 
[6]  Hatakka, A., and K. E. Hammel, “Fungal Biodegradation of Lignocelluloses,” Industrial Applications, M. Hofrichter, ed., pp. 319-340, Berlin, Heidelberg: Springer Berlin Heidelberg, 2011.
 
[7]  Marco-Urrea, E., and C. A. Reddy, “Degradation of Chloro-organic Pollutants by White Rot Fungi,” Microbial Degradation of Xenobiotics, N. S. Singh, ed., pp. 31-66, Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.
 
[8]  Lundell, T., “Ligninolytic System of the White-rot Fungus Phlebia radiata: Lignin Model Compound Studies,” Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, 1993.
 
[9]  Karhunen, E., A. Kantelinen, and M.-L. Niku-Paavola, “Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata,” Archives of Biochemistry and Biophysics, 279 (1). 25-31, 1990.
 
[10]  Moilanen, A. M., T. Lundell, T. Vares, and A. Hatakka, “Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata,” Applied Microbiology and Biotechnology, 45 (6). 792-799, 1996.
 
[11]  Hildén, K. S., M. R. Mäkelä, T. K. Hakala, A. Hatakka, and T. Lundell, “Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata,” Current Genetics, 49 (2). 97-105, 2005.
 
[12]  Lundell, T. K., M. R. Mäkelä, and K. Hildén, “Lignin-modifying enzymes in filamentous basidiomycetes – ecological, functional and phylogenetic review,” Journal of Basic Microbiology, 50 (1). 5-20, 2010.
 
[13]  Hofrichter, M., R. Ullrich, M. J. Pecyna, C. Liers, and T. Lundell, “New and classic families of secreted fungal heme peroxidases,” Applied Microbiology and Biotechnology, 87 (3). 871-897, 2010.
 
[14]  Glenn, J. K., and M. H. Gold, “Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium,” Archives of Biochemistry and Biophysics, 242 (2). 329-341, 1985.
 
[15]  Paszczyński, A., V.-B. Huynh, and R. Crawford, “Enzymatic activities of an extracellular, manganese-dependent peroxidase from Phanerochaete chrysosporium,” FEMS Microbiology Letters, 29 (1-2). 37-41, 1985.
 
[16]  Hofrichter, M., “Review: lignin conversion by manganese peroxidase (MnP),” Enzyme and Microbial Technology, 30 (4). 454-466, 2002.
 
[17]  Wariishi, H., K. Valli, and M. H. Gold, “Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators,” Journal of Biological Chemistry, 267 (33). 23688-23695, 1992.
 
[18]  Kirk, T. K., and R. L. Farrell, “Enzymatic “Combustion”: The Microbial Degradation of Lignin,” Annual Review of Microbiology, 41 (1). 465-501, 1987.
 
[19]  Kersten, P., and D. Cullen, “Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium,” Fungal Genetics and Biology, 44 (2). 77-87, 2007.
 
[20]  Buswell, J. A., E. Odier, and T. K. Kirk, “Lignin Biodegradation,” Critical Reviews in Biotechnology, 6 (1). 1-60, 1987.
 
[21]  Martinez, D., L. F. Larrondo, N. Putnam, M. D. S. Gelpke, K. Huang, J. Chapman, K. G. Helfenbein, P. Ramaiya, J. C. Detter, F. Larimer, P. M. Coutinho, B. Henrissat, R. Berka, D. Cullen, and D. Rokhsar, “Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78,” Nat Biotech, 22 (6). 695-700, 2004.
 
[22]  Hildén, K., A. T. Martinez, A. Hatakka, and T. Lundell, “The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent,” Fungal Genetics and Biology, 42 (5). 403-419, 2005.
 
[23]  Martı́nez, A. T., “Molecular biology and structure-function of lignin-degrading heme peroxidases,” Enzyme and Microbial Technology, 30 (4). 425-444, 2002.
 
[24]  Sundaramoorthy, M., K. Kishi, M. H. Gold, and T. L. Poulos, “Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase,” Journal of Biological Chemistry, 272 (28). 17574-17580, 1997.
 
[25]  Kusters-van Someren, M., K. Kishi, T. Lundell, and M. H. Gold, “The Manganese Binding Site of Manganese Peroxidase: Characterization of an Asp179Asn Site-Directed Mutant Protein,” Biochemistry, 34 (33). 10620-10627, 1995.
 
[26]  Kishi, K., M. Kusters-van Someren, M. B. Mayfield, J. Sun, T. M. Loehr, and M. H. Gold, “Characterization of Manganese (II) Binding Site Mutants of Manganese Peroxidase,” Biochemistry, 35 (27). 8986-8994, 1996.
 
[27]  Boucher, L. J., K. Koeber, M. Kotowski, and D. Tille, “Coordination Compounds of Manganese,” Coordination Compounds 7, H. Demmer, M. Kotowski, E. Schleitzer-Rust and D. Tille, eds., pp. 1-2, Berlin, Heidelberg: Springer Berlin Heidelberg, 1989.
 
[28]  Sundaramoorthy, M., K. Kishi, M. H. Gold, and T. L. Poulos, “The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution,” Journal of Biological Chemistry, 269 (52). 32759-32767, 1994.
 
[29]  Sundaramoorthy, M., H. L. Youngs, M. H. Gold, and T. L. Poulos, “High-Resolution Crystal Structure of Manganese Peroxidase:  Substrate and Inhibitor Complexes,” Biochemistry, 44 (17). 6463-6470, 2005.
 
[30]  Gold, M. H., H. L. Youngs, and M. D. Sollewijn Gelpke, “Manganese Peroxidase,” Metal Ions in Biological Systems: Volume 37: Manganese and Its Role in Biological Processes, A. Sigel and H. Sigel, eds., pp. 559-586, New York, USA: CRC Press, 2000.
 
[31]  Kuan, I. C., K. A. Johnson, and M. Tien, “Kinetic analysis of manganese peroxidase. The reaction with manganese complexes,” Journal of Biological Chemistry, 268 (27). 20064-20070, 1993.
 
[32]  Kishi, K., H. Wariishi, L. Marquez, H. B. Dunford, and M. H. Gold, “Mechanism of Manganese Peroxidase Compound II Reduction. Effect of Organic Acid Chelators and pH,” Biochemistry, 33 (29). 8694-8701, 1994.
 
[33]  Paszczyński, A., V.-B. Huynh, and R. Crawford, “Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium,” Archives of Biochemistry and Biophysics, 244 (2). 750-765, 1986.
 
[34]  Wariishi, H., H. B. Dunford, I. D. MacDonald, and M. H. Gold, “Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism,” Journal of Biological Chemistry, 264 (6). 3335-3340, 1989.
 
[35]  Gregory, D. S., A. C. R. Martin, J. C. Cheetham, and A. R. Rees, “The prediction and characterization of metal binding sites in proteins,” Protein Engineering, 6 (1). 29-35, 1993.
 
[36]  Kennedy, M. L., and B. R. Gibney, “Metalloprotein and redox protein design,” Current Opinion in Structural Biology, 11 (4). 485-490, 2001.
 
[37]  Maneiro, M., W. F. Ruettinger, E. Bourles, G. L. McLendon, and G. C. Dismukes, “Kinetics of proton-coupled electron-transfer reactions to the manganese-oxo “cubane” complexes containing the Mn4O and Mn4O core types,” Proceedings of the National Academy of Sciences, 100 (7). 3707-3712, 2003.
 
[38]  Garcia, J. S., C. S. d. Magalhães, and M. A. Z. Arruda, “Trends in metal-binding and metalloprotein analysis,” Talanta, 69 (1). 1-15, 2006.
 
[39]  Pecoraro, V., and W. Hsieh, “The use of model complexes to elucidate the structure and function of manganese redox enzymes,” Metals in Biological Systems, A. S. a. H. Sigel, ed., pp. 429-504, New York, USA: CRC Press, 2000.
 
[40]  Puglisi, A., G. Tabbı̀, and G. Vecchio, “Bioconjugates of cyclodextrins of manganese salen-type ligand with superoxide dismutase activity,” Journal of Inorganic Biochemistry, 98 (6). 969-976, 2004.
 
[41]  Childs, R. E., and W. G. Bardsley, “The steady-state kinetics of peroxidase with 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen,” Biochemical Journal, 145 (1). 93-103, 1975.
 
[42]  Ruiz-Dueñas, F. J., M. Morales, M. Pérez-Boada, T. Choinowski, M. J. Martínez, K. Piontek, and Á. T. Martínez, “Manganese Oxidation Site in Pleurotus eryngii Versatile Peroxidase:  A Site-Directed Mutagenesis, Kinetic, and Crystallographic Study,” Biochemistry, 46 (1). 66-77, 2007.
 
[43]  Cleland, W. W., “1 Steady State Kinetics,” The Enzymes, D. B. Paul, ed., pp. 1-65: Academic Press, 1970.
 
[44]  Sandler, M., and H. J. Smith, “Introduction to the use of enzyme inhibitors as drugs,” Design of Enzyme Inhibitors as Drugs, , M. Sandler and H. J. Smith, eds., pp. 1-18, Oxford, UK: Oxford University Press, 1989.
 
[45]  Blake, R. D., Informational biopolymers of genes and gene expression, Sausalito, CA: University Science, 2004.
 
[46]  Millero, F. J., Chemical Oceanography, Fourth Edition, Boca Raton: CRC Press, 2013.
 
[47]  Sarkar, S., A. T. Martı́nez, and M. a. J. Martı́nez, “Biochemical and molecular characterization of a manganese peroxidase isoenzyme from Pleurotus ostreatus,” Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1339 (1). 23-30, 1997.
 
[48]  Liu, C., and H. Xu, “The metal site as a template for the metalloprotein structure formation,” Journal of Inorganic Biochemistry, 88 (1). 77-86, 2002.
 
[49]  Emeléus, H. J., and J. S. Anderson, “Modern Aspects of Inorganic Chemistry,” 1956.
 
[50]  Arnold, F. H., and J.-H. Zhang, “Metal-mediated protein stabilization,” Trends in Biotechnology, 12 (5). 189-192, 1994.
 
[51]  Ufot, U. F., and M. I. Akpanabiatu, “An engineered Phlebia radiata manganese peroxidase: expression, refolding, purification and preliminary characterization,” American Journal of Molecular Biology, 2 (4). 359-370, 2012.
 
[52]  Ite, A. E., I. I. Udousoro, and U. J. Ibok, “Distribution of Some Atmospheric Heavy Metals in Lichen and Moss Samples Collected from Eket and Ibeno Local Government Areas of Akwa Ibom State, Nigeria,” American Journal of Environmental Protection, 2 (1). 22-31, 2014.
 
[53]  Ite, A. E., N. F. Hanney, and K. T. Semple, “The Effect of Hydroxycinnamic Acids on the Microbial Mineralisation of Phenanthrene in Soil,” International Journal of Environmental Bioremediation & Biodegradation, 3 (2). 40-47, 2015.
 
[54]  Ufot, U. F., “Expression and Characterisation of a Novel Manganese Peroxidise from Phlebia radiata,” Department of Biochemistry, University of Sussex, University of Sussex, Brighton, United Kingdom, 2010.
 
[55]  Johnson, F., G. H. Loew, and P. Du, “Prediction of Mn(II) binding site of manganese peroxidase from homotology modeling,” Plant peroxidases: Biochemistry and Physiology: III International Symposium 1993: proceedings, xiii, 497 p., K. G. Weirder, S. K. Rasmussen, C. Penel and H. Greppin, eds., pp. 31 – 34, Geneva, Switzerland: University of Copenhagen and University of Geneva, 1993.
 
[56]  Harris, R. Z., H. Wariishi, M. H. Gold, and P. R. Ortiz de Montellano, “The catalytic site of manganese peroxidase. Regiospecific addition of sodium azide and alkylhydrazines to the heme group,” Journal of Biological Chemistry, 266 (14). 8751-8758, 1991.
 
[57]  Whitwam, R. E., K. R. Brown, M. Musick, M. J. Natan, and M. Tien, “Mutagenesis of the Mn2+-Binding Site of Manganese Peroxidase Affects Oxidation of Mn2+ by both Compound I and Compound II,” Biochemistry, 36 (32). 9766-9773, 1997.
 
[58]  Da Silva, J. F., and R. J. P. Williams, The Biological Chemistry of the Elements: the Inorganic Chemistry of Life: Oxford University Press, 2001.
 
[59]  Sunda, W. G., and S. A. Huntsman, “Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira,” Limnology and Oceanography, 28 (5). 924-934, 1983.
 
[60]  Sunda, W. G., and S. A. Huntsman, “Relationships among growth rate, cellular manganese and manganese transport kinetics in estuarine and oceanic species of the diatom Thalassiosira,” Journal of Phycology, 22 (3). 259-270, 1986.
 
[61]  Sunda, W. G., “Trace Metal Interactions with Marine Phytoplankton,” Biological Oceanography, 6 (5-6). 411-442, 1989.
 
[62]  Coolbear, T., J. M. Whittaker, and R. M. Daniel, “The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1,” Biochemical Journal, 287 (Pt 2). 367-374, 1992.
 
[63]  Nieboer, E., and D. H. S. Richardson, “The replacement of the nondescript term ‘heavy metals’ by a biologically and chemically significant classification of metal ions,” Environmental Pollution Series B, Chemical and Physical, 1 (1). 3-26, 1980.
 
[64]  Haas, K. L., and K. J. Franz, “Application of Metal Coordination Chemistry to Explore and Manipulate Cell Biology,” Chemical Reviews, 109 (10). 4921-4960, 2009.