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Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase.

Wilcox SK, Putnam CD, Sastry M, Blankenship J, Chazin WJ, McRee DE, Goodin DB
Biochemistry 37 16853-62 (1998)
Cited: 22 times
EuropePMC logo PMID: 9836578

Abstract

The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

Reviews citing this publication (5)

  1. Protein design: toward functional metalloenzymes. Yu F, Cangelosi VM, Zastrow ML, Tegoni M, Plegaria JS, Tebo AG, Mocny CS, Ruckthong L, Qayyum H, Pecoraro VL. Chem Rev 114 3495-3578 (2014)
  2. Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases. Ruiz-Dueñas FJ, Morales M, García E, Miki Y, Martínez MJ, Martínez AT. J Exp Bot 60 441-452 (2009)
  3. Tailoring new enzyme functions by rational redesign. Cedrone F, Ménez A, Quéméneur E. Curr Opin Struct Biol 10 405-410 (2000)
  4. Biosynthetic inorganic chemistry. Lu Y. Angew Chem Int Ed Engl 45 5588-5601 (2006)
  5. Construction of heme enzymes: four approaches. Watanabe Y. Curr Opin Chem Biol 6 208-216 (2002)

Articles citing this publication (17)

  1. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Padilla JE, Colovos C, Yeates TO. Proc Natl Acad Sci U S A 98 2217-2221 (2001)
  2. Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study. Bonomo RP, Imperllizzeri G, Pappalardo G, Rizzarelli E, Tabbì G. Chemistry 6 4195-4202 (2000)
  3. Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase. Smith AT, Doyle WA, Dorlet P, Ivancich A. Proc Natl Acad Sci U S A 106 16084-16089 (2009)
  4. Biosynthesis of artificial microperoxidases by exploiting the secretion and cytochrome c maturation apparatuses of Escherichia coli. Braun M, Thöny-Meyer L. Proc Natl Acad Sci U S A 101 12830-12835 (2004)
  5. Metalloprotein and metallo-DNA/RNAzyme design: current approaches, success measures, and future challenges. Lu Y. Inorg Chem 45 9930-9940 (2006)
  6. Lanthanide-binding helix-turn-helix peptides: solution structure of a designed metallonuclease. Welch JT, Kearney WR, Franklin SJ. Proc Natl Acad Sci U S A 100 3725-3730 (2003)
  7. Changing the substrate specificity of cytochrome c peroxidase using directed evolution. Iffland A, Gendreizig S, Tafelmeyer P, Johnsson K. Biochem Biophys Res Commun 286 126-132 (2001)
  8. Enantiomeric discrimination of Ru-substrates by cytochrome P450cam. Dmochowski IJ, Winkler JR, Gray HB. J Inorg Biochem 81 221-228 (2000)
  9. NMR study of manganese(II) binding by a new versatile peroxidase from the white-rot fungus Pleurotus eryngii. Banci L, Camarero S, Martínez AT, Martínez MJ, Pérez-Boada M, Pierattelli R, Ruiz-Dueñas FJ. J Biol Inorg Chem 8 751-760 (2003)
  10. Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase. Pfister TD, Mirarefi AY, Gengenbach AJ, Zhao X, Danstrom C, Conatser N, Gao YG, Robinson H, Zukoski CF, Wang AH, Lu Y. J Biol Inorg Chem 12 126-137 (2007)
  11. Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin. Hunter CL, Maurus R, Mauk MR, Lee H, Raven EL, Tong H, Nguyen N, Smith M, Brayer GD, Mauk AG. Proc Natl Acad Sci U S A 100 3647-3652 (2003)
  12. Enhancing Mn(II)-Binding and Manganese Peroxidase Activity in a Designed Cytochrome c Peroxidase through Fine-Tuning Secondary-Sphere Interactions. Hosseinzadeh P, Mirts EN, Pfister TD, Gao YG, Mayne C, Robinson H, Tajkhorshid E, Lu Y. Biochemistry 55 1494-1502 (2016)
  13. Surface grafting onto template-assembled synthetic protein scaffolds in molecular recognition. Fernandez-Carneado J, Grell D, Durieux P, Hauert J, Kovacsovics T, Tuchscherer G. Biopolymers 55 451-458 (2000)
  14. Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes. Teske JG, Savenkova MI, Mauro JM, Erman JE, Satterlee JD. Protein Expr Purif 19 139-147 (2000)
  15. Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase. Feng M, Tachikawa H, Wang X, Pfister TD, Gengenbach AJ, Lu Y. J Biol Inorg Chem 8 699-706 (2003)
  16. Design of Heteronuclear Metalloenzymes. Bhagi-Damodaran A, Hosseinzadeh P, Mirts E, Reed J, Petrik ID, Lu Y. Methods Enzymol 580 501-537 (2016)
  17. MnII is not a productive substrate for wild-type or recombinant lignin peroxidase isozyme H2. Sollewijn Gelpke MD, Sheng D, Gold MH. Arch Biochem Biophys 381 16-24 (2000)


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