Structural and spectroscopic characterisation of a heme peroxidase from sorghum |
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Authors: | Chukwudi I. Nnamchi Gary Parkin Igor Efimov Jaswir Basran Hanna Kwon Dimitri A. Svistunenko Jon Agirre Bartholomew N. Okolo Anene Moneke Bennett C. Nwanguma Peter C. E. Moody Emma L. Raven |
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Affiliation: | 1.Department of Microbiology,University of Nigeria,Nsukka,Nigeria;2.Department of Chemistry,University of Leicester,Leicester,UK;3.Department of Molecular and Cell Biology, Henry Wellcome Laboratory for Structural Biology,University of Leicester,Leicester,UK;4.School of Biological Sciences,University of Essex,Colchester,UK;5.York Structural Biology Laboratory, Department of Chemistry,The University of York,York,UK;6.Department of Biochemistry,University of Nigeria,Nsukka,Nigeria |
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Abstract: | A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV–visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A FeIII/FeII reduction potential of ?266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na+ ion) and proximal (assigned as a Ca2+) sides of the heme, which is consistent with the Ca2+-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions. |
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