NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.
Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.
Veitch
N C
NC
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, England.
Tams
J W
JW
Vind
J
J
Dalbøge
H
H
Welinder
K G
KG
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D003302:Coprinus / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005973:Glutamine; D006418:Heme; D010649:Phenylalanine; D001216:Asparagine; D009195:Peroxidase