Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase

Document Type : Original article

Authors

1 Biophysics and Computational Biology Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran

2 Institute of Biotechnology, Shiraz University, Shiraz, Iran

Abstract

Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these substitutions, the number of stable hydrogen bonds and α-helical content of HRP are increased and the proximal Ca2+ binding pocket becomes more integrated. The results revealed that although Glu174-heme hydrogen bond is lost after mutation, formation of a new hydrogen bonding network contributes to the stability of heme-protein linkage. Together, it may be concluded that these substitutions enhance the stability of the protein moiety as well as the heme-protein non-covalent interactions. In the enzyme active site, we observed increased accessibility of peroxide binding site and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the bottleneck entry of the peroxide-binding site has become wider and more flexible upon substitutions. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is more extended in mutated enzyme. These observations suggest that the reactivity of the enzyme to its substrates has increased. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved upon manipulation of charge distribution on protein surface.

Keywords

References

1. Colonna S, Gaggero N, Richelmi C, Pasta P. Recent biotechnological developments in the use of peroxidases. Trends Biotechnol 1999;17:163-168.
2. Hamid M, Khalil ur R. Potential applications of peroxidases. Food Chem 2009;115:1177-1186.
3. Azevedo AM, Martins VC, Prazeres DMF, Vojinovic V, Cabral JMS, Fonseca LP. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol Annu Rev 2003;9:199-247.
4. Raghu P, Reddy TM, Reddaiah K, Jaidev L, Narasimha G. A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly (l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone. Enzyme Microb Technol 2013;52:377-385.
5. Liu X, Luo L, Ding Y, Xu Y. Amperometric biosensors based on alumina nanoparticles-chitosan-horseradish peroxidase nanobiocomposites for the determination of phenolic compounds. Analyst 2011;136:696-701.
6. Kafi A, Wu G, Chen A. A novel hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto Au-modified titanium dioxide nanotube arrays. Biosens Bioelectron 2008;24:566-571.
7. Ugarova NN, Rozhkova GD, Berezin IV. Chemical modification of the ε-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme. Biochim Biophys Acta 1979;570:31-42.
8. Miland E, Smyth MR, Ó'Fágáin C. Increased thermal and solvent tolerance of acetylated horseradish peroxidase. Enzyme Microb Technol 1996;19:63-67.
9. Miland E, Smyth MR, Ó'Fágáin C. Modification of horseradish peroxidase with bifunctional N-hydroxysuccinimide esters: effects on molecular stability. Enzyme Microb Technol 1996;19:242-249.
10. O’Brien AM, Ó'Fágáin C, Nielsen PF, Welinder KG. Location of crosslinks in chemically stabilized horseradish peroxidase. Implications for design of crosslinks. Biotechnol Bioeng 2001;76:277-284.
11. O'Brien AM, Smith AT, Ó'Fágáin C. Effects of phthalic anhydride modification on horseradish peroxidase stability and activity. Biotechnol Bioeng 2003;81:233-240.
12. Song HY, Yao JH, Liu JZ, Zhou SJ, Xiong YH, Ji LN. Effects of phthalic anhydride modification on horseradish peroxidase stability and structure. Enzyme Microb Technol 2005;36:605-611.
13. Liu JZ, Wang TL, Huang MT, Song HY, Weng LP, Ji LN. Increased thermal and organic solvent tolerance of modified horseradish peroxidase. Protein Eng Des Sel 2006;19:169-173.
14. Ryan BJ, Ó'Fágáin C. Effects of mutations in the helix G region of horseradish peroxidase. Biochimie 2008;90:1414–1421.
15. Mogharrab N, Ghourchian H. Anthraquinone 2-carboxylic acid as an electron shuttling mediator and attached electron relay for horseradish peroxidase. Electrochem Commun 2005;7:466-471.
16. Mogharrab N, Ghourchian H, Amininasab M. Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation. Biophys J 2007;92:1192-1203.
17. Urrutigoity M, Baboulene M, Lattes A. Use of pyrocarbonates for chemical modification of histidine residues of horseradish peroxidase. Bioorg Chem 1991;19:66-76.
18. O’Brien AM. Chemical modification and characterization of horseradish peroxidase and its derivatives for environmental applications. Ph.D. Thesis. Dublin City University: Ireland 1997.
19. Khajeh K, Naderi-Manesh H, Ranjbar B, Moosavi-Movahedi AA, Nemat-Gorgani M. Chemical modification of lysine residues in Bacillus α-amylases: effect on activity and stability. Enzyme Microb Technol 2001;28:543-549.
20. Moreno JM, Ó'Fágáin C. Activity and stability of native and modified alanine aminotransferase in cosolvent systems and denaturants. J Mol Catal B Enzym 1997;2:271-279.
21. Elsner C, Grahn S, Bauer S, Ullmann D, Kurth T, Jakubke HD. Effects of chemical modification of lysine residues in trypsin. J Mol Catal B: Enzym 2000;8:193-200.
22. Szabó A, Kotormán M, Laczkó I, Simon LM. Improved stability and catalytic activity of chemically modified papain in aqueous organic solvents. Process Biochem 2009;44:199-204.
23. Xue Y, Wu CY, Branford-White CJ, Ning X, Nie HL, Zhu LM. Chemical modification of stem bromelain with anhydride groups to enhance its stability and catalytic activity. J Mol Catal B Enzym 2010;63:188-193.
24. Welinder KG. Amino acid sequence studies of horseradish peroxidase. Eur J Biochem 1979;95:483-502.
25. Yang BY, Gray JSS, Montgomery R. The glycans of horseradish peroxidase. Carbohydr Res 1996;287:203-212.
26. Navapour L, Mogharrab N, Amininasab M. How modification of accessible lysines to phenylalanine modulates the structural and functional properties of horseradish peroxidase: a simulation study. PLoS One 2014;9:e109062.
27. Gajhede M, Schuller DJ, Henriksen A, Smith AT, Poulos TL. Crystal structure of horseradish peroxidase C at 2.15 Å resolution. Nat Struct Biol 1997;4:1032-1038.
28. van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC. GROMACS: fast, flexible, and free. J Comput Chem 2005;26:1701-1718.
29. Oostenbrink C, Villa A, Mark AE, van Gunsteren WF. A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem 2004;25:1656-1676.
30. Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys 2007;126:014101.
31. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular-dynamics with coupling to an external bath. J Chem Phys 1984;81:3684-3690.
32. Parrinello M, Rahman A. Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys 1981;52:7182-7190.
33. Hess B, Bekker H, Berendsen HJC, Fraaije JGEM. LINCS: a linear constraint solver for molecular simulations. J Comput Chem 1997;18:1463-1472.
34. Darden T, York D, Pedersen L. Particle mesh Ewald: an N Log (N) method for Ewald sums in large systems. J Chem Phys 1993;98:1463-1472.
35. Yin S, Ding F, Dokholyan NV. Eris: an automated estimator of protein stability. Nat Meth 2007;4:466-467.
36. Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983;22:2577-2637.
37. Veitch NC. Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 2004;65:249-259.
38. Khajehpour M, Rietveld I, Vinogradov S, Prabhu NV, Sharp KA, Vanderkooi JM. Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase. Proteins Struct Funct Genet 2003;53:656-666.
39. Poulos TL, Kraut J. The stereochemistry of peroxidase catalysis. J Biol Chem 1980;255:8199-8205.
40. Savenkova MI, Newmyer SL, Ortiz de Montellano PR. Rescue of His-42→Ala horseradish peroxidase by a Phe-41→His mutation: engineering of a surrogate catalytic histidine. J Biol Chem 1996;271:24598-24603.
41. Gajhede M. Plant peroxidases: substrate complexes with mechanistic implications. Biochem Soc Trans 2001;29:91-99.
42. Henriksen A, Schuller DJ, Meno K, Welinder KG, Smith AT, Gajhede M. Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by x-ray crystallography. Biochemistry 1998;37:8054-8060.
43. Ryan O, Smyth MR, Ó'Fágáin C. Thermostabilized chemical derivatives of horseradish peroxidease. Enzyme Microb Technol 1994;16:501-505.
44. Liu JZ, Song HY, Weng LP, Ji LN. Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase. J Mol Catal B: Enzym 2002;18:225-232.
45. Hassani L. Chemical modification of horseradish peroxidase with carboxylic anhydrides: effect of negative charge and hydrophilicity of the modifiers on thermal stability. J Mol Catal B: Enzym 2012;80:15-19.
46. Ryan BJ, Ó'Fágáin C. Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide. Biochimie 2007;89:1029-1032.
47. Ryan B. Site directed mutagenesis studies of horseradish peroxidase. Ph.D. Thesis. Dublin City University:Ireland 2006.
48. Howes BD, Feis A, Raimondi L, Indiani C, Smulevich G. The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase. J Biol Chem 2001;276:40704-40711.
Molecular Biology Research Communications
Volume 5, Issue 3 - Serial Number 3
September 2016
Pages 175-192

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