1 14s BiochemicalSocietyTransactions(1 992) 20 Investigation of native and mutant plant peroxidascs by NMR
NIGEL c. VEITCH.. ROBERT J.P. WILLIAMS.. A N D M T.SMITH, STEVEN A. SANDERS, ROGEFt N.F. THO-, ROBERT C. BRAY and JULIAN F. BURKE.
The N M R spectra of the cyanide-ligated plant cnzyme and F41V mutant arc illustrated by the data given in Fig. 2. Note that cyanide binds to the vacant sixth coordination site of resting state peroxidase altering the spin state from predominantly high spin to low spin. Assignments of hacmlinked resonances were made using both one and twod i m e n s i d nuclear Ovexhauscr enhancement experiments.
* w c chemistry ~aboratory.University of oxford.south Parks R o d , Oxford, OX1 3QR. U.K. and Biochemistry Laboratory. Univasity of Sussex, Brighton. BN19QG. U.K.. A central aim of structural studies on plant pcroxidascs must be to uncover the functional significance of residues which arc critical for both peroxide activation and binding of aromatic donor molecules, key events during catalytic turnover. However crystallographic data is limited, with a low resolution structure of a single horseradish peroxidase isocnzyme (HRP E5) only recently available [l]. Correspondingly many other spectroscopic techniques have been employed although of these only NMR spectrosc~pyhas the potential to directly probe the structural aspects of pcroxidase function. Recent applications of the latter have included studies of the haem binding pocket and aromatic donor molecule binding site of horseradish peroxidase isocnzyme C (HRP C) by both one and two dimensional experimental methods [2-61. From the viewpoint of NMR however. HRP C is a complex system with its relatively high molecular mass (42kDa). paramagnetic properties and 18% carbohydrate content. Hence the recent expression of a synthetic gene encoding HRP C in E.coli and subsequent production of site-directed mutants is of great importance for structural studies of this cnzyme utilising NMR [7]. Here we present preliminary data for the characterisation of the horseradish peroxidase isocnzyme C mutant. F41V. The residue Phe 41 is completely conserved in all the plant peroxidases so far sequenced. Alignment of thew sequences with the amino acid sequence of yeast cytochrome c peroxidase indicates that the residue is analogous to Trp 51 in distal helix B 181. A recent crystallographic analysis of the cytochrome c peroxidasc mutant, W51F. concluded that the Phe residue was situated approximately 3.3A above the haem plane and that WSlF was a good structural model for the region containing distal helix B (residues 32-43) in horseradish pcroxidase [9]. The probable relationship of Phe 41 to the haem group and to the two axial histidine residues is shown in Fig. 1.
PIN 41
Fig. 1: Representation of the haem group and associated residues in HRP C. The numbering of haem substituents is also given. The recombinant horseradish peroxidase mutant, produced in the form of inclusion bodies was solubilised. folded and activated essentially according to the protocols originally established for the recombinant peroxidase HRP C [71. Approximately 14mg of HRP C :F41V peroxidase was purified for NMR studies with a final R.Z. value of 3.8. Onedimensional spectra were recorded on a 500 MHz Bruker spectrometer over 4K data points with a spectral width of 30 kHz. 1600 transients were collected in each case. Chemical shift measurements were as previously described [4].
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Fig. 2: Comparison of the h y p e d n e shifted resonances of cyanide-ligated plant HRP C and HRP C':F41V mutant. The spectra were recorded at 30'C in 2OmM KH,PO *15mM KCND20 at pH 7.6 with a protein concentration of O.&nM. Examination of the entire NMR spectrum of the HRP C*:F41V mutant indicates that the overall fold of the protein is identical to that of the plant enzyme. However it is clear from Fig.2 that there are significant differences between the spectra of plant and mutant pcroxidase associated with the haem-linked region. most notably the large upfield shifts of the 8-CH3 and 4-H resonances. The pattern of these shifts is unusual and mayaindicate that in addition to the local perturbation resulting from a Phe to Val substitution the electronic properties of the cyanide-ligated mutant peroxidase differ from those of the plant peroxidasc. This work was supported by the Science and Engineering Research Council and Merton College, Oxford. 1. Morita, Y., Mikami. B.. Yamashita. H.. Lee, J.Y.. Aibara. S.. Sam, M., Katsube, Y. & Tanaka N. (1991) in Molecular and Physiological Aspects of Plant Peroxidases (tobarzewski. J.. Greppin. H..Penel, C. & Gaspar. T.eds.). University of Geneva. Switzerland 2. Sakurada, J.. Takahashi. S. & Hosoya, T. (1986) J. Biol. Chem. 261,9657-9662 3. Thanabal, V.. De Ropp. J.S. & L a Mar, G.N. (1987) J. Am. Chem. Soc. 109.7516-7525 4. Veitch. N.C. & Williams, R.J.P. (1990) Eur. J. Biochem. 189,351-362 5. Veitch. N.C. & Williams, R.J.P. (1991) in Molecular and Physiological Aspects of Plant Peroxidases (tobarzewski. J.. Greppin. H.,Penel. C. & Gaspar. T. eds.). University of Geneva, Switzerland 6. De Ropp. J.S., Yu. L.P. & La Mar, G.N. (1991) J. Biomolecular NMR 1, 175-190 7. Smith, A.T., Santama, N.. Dacey. S..Edwards. M.. Bray, R.C.. Thomeley. R.N.F. & Burke J.F. (1990) J. Biol. Chem. 265,13335-13343 8. Welinder, K.G. (1985) Eur. J. Biochem. 151.415-424 9. Wang, J.. Mauro. J.M.. Edwards. S.L..Oatley, S.J., Fishel. L.A., Ashford, V.A., Xuong, N. & Kraut, J. (1990) Biochemistry 29.7 160-7 173
NIGEL c. VEITCH.. ROBERT J.P. WILLIAMS.. A N D M T.SMITH, STEVEN A. SANDERS, ROGEFt N.F. THO-, ROBERT C. BRAY and JULIAN F. BURKE.
The N M R spectra of the cyanide-ligated plant cnzyme and F41V mutant arc illustrated by the data given in Fig. 2. Note that cyanide binds to the vacant sixth coordination site of resting state peroxidase altering the spin state from predominantly high spin to low spin. Assignments of hacmlinked resonances were made using both one and twod i m e n s i d nuclear Ovexhauscr enhancement experiments.
* w c chemistry ~aboratory.University of oxford.south Parks R o d , Oxford, OX1 3QR. U.K. and Biochemistry Laboratory. Univasity of Sussex, Brighton. BN19QG. U.K.. A central aim of structural studies on plant pcroxidascs must be to uncover the functional significance of residues which arc critical for both peroxide activation and binding of aromatic donor molecules, key events during catalytic turnover. However crystallographic data is limited, with a low resolution structure of a single horseradish peroxidase isocnzyme (HRP E5) only recently available [l]. Correspondingly many other spectroscopic techniques have been employed although of these only NMR spectrosc~pyhas the potential to directly probe the structural aspects of pcroxidase function. Recent applications of the latter have included studies of the haem binding pocket and aromatic donor molecule binding site of horseradish peroxidase isocnzyme C (HRP C) by both one and two dimensional experimental methods [2-61. From the viewpoint of NMR however. HRP C is a complex system with its relatively high molecular mass (42kDa). paramagnetic properties and 18% carbohydrate content. Hence the recent expression of a synthetic gene encoding HRP C in E.coli and subsequent production of site-directed mutants is of great importance for structural studies of this cnzyme utilising NMR [7]. Here we present preliminary data for the characterisation of the horseradish peroxidase isocnzyme C mutant. F41V. The residue Phe 41 is completely conserved in all the plant peroxidases so far sequenced. Alignment of thew sequences with the amino acid sequence of yeast cytochrome c peroxidase indicates that the residue is analogous to Trp 51 in distal helix B 181. A recent crystallographic analysis of the cytochrome c peroxidasc mutant, W51F. concluded that the Phe residue was situated approximately 3.3A above the haem plane and that WSlF was a good structural model for the region containing distal helix B (residues 32-43) in horseradish pcroxidase [9]. The probable relationship of Phe 41 to the haem group and to the two axial histidine residues is shown in Fig. 1.
PIN 41
Fig. 1: Representation of the haem group and associated residues in HRP C. The numbering of haem substituents is also given. The recombinant horseradish peroxidase mutant, produced in the form of inclusion bodies was solubilised. folded and activated essentially according to the protocols originally established for the recombinant peroxidase HRP C [71. Approximately 14mg of HRP C :F41V peroxidase was purified for NMR studies with a final R.Z. value of 3.8. Onedimensional spectra were recorded on a 500 MHz Bruker spectrometer over 4K data points with a spectral width of 30 kHz. 1600 transients were collected in each case. Chemical shift measurements were as previously described [4].
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Fig. 2: Comparison of the h y p e d n e shifted resonances of cyanide-ligated plant HRP C and HRP C':F41V mutant. The spectra were recorded at 30'C in 2OmM KH,PO *15mM KCND20 at pH 7.6 with a protein concentration of O.&nM. Examination of the entire NMR spectrum of the HRP C*:F41V mutant indicates that the overall fold of the protein is identical to that of the plant enzyme. However it is clear from Fig.2 that there are significant differences between the spectra of plant and mutant pcroxidase associated with the haem-linked region. most notably the large upfield shifts of the 8-CH3 and 4-H resonances. The pattern of these shifts is unusual and mayaindicate that in addition to the local perturbation resulting from a Phe to Val substitution the electronic properties of the cyanide-ligated mutant peroxidase differ from those of the plant peroxidasc. This work was supported by the Science and Engineering Research Council and Merton College, Oxford. 1. Morita, Y., Mikami. B.. Yamashita. H.. Lee, J.Y.. Aibara. S.. Sam, M., Katsube, Y. & Tanaka N. (1991) in Molecular and Physiological Aspects of Plant Peroxidases (tobarzewski. J.. Greppin. H..Penel, C. & Gaspar. T.eds.). University of Geneva. Switzerland 2. Sakurada, J.. Takahashi. S. & Hosoya, T. (1986) J. Biol. Chem. 261,9657-9662 3. Thanabal, V.. De Ropp. J.S. & L a Mar, G.N. (1987) J. Am. Chem. Soc. 109.7516-7525 4. Veitch. N.C. & Williams, R.J.P. (1990) Eur. J. Biochem. 189,351-362 5. Veitch. N.C. & Williams, R.J.P. (1991) in Molecular and Physiological Aspects of Plant Peroxidases (tobarzewski. J.. Greppin. H.,Penel. C. & Gaspar. T. eds.). University of Geneva, Switzerland 6. De Ropp. J.S., Yu. L.P. & La Mar, G.N. (1991) J. Biomolecular NMR 1, 175-190 7. Smith, A.T., Santama, N.. Dacey. S..Edwards. M.. Bray, R.C.. Thomeley. R.N.F. & Burke J.F. (1990) J. Biol. Chem. 265,13335-13343 8. Welinder, K.G. (1985) Eur. J. Biochem. 151.415-424 9. Wang, J.. Mauro. J.M.. Edwards. S.L..Oatley, S.J., Fishel. L.A., Ashford, V.A., Xuong, N. & Kraut, J. (1990) Biochemistry 29.7 160-7 173
