NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants

Abstract

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 andlor 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 Phel56 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 peroxidasesThe frequency with which peroxidase enzymes are being isolated and characterised from new sources far outweighs progress in our knowledge both of their three-dimensional structures and of their interactions with substrate and ligand molecules. A peroxidase in this category is that recently isolated and characterised from the ink cap basidiomycete Co-

Pentacoordination of the heme iron of Arthromyces ramosus peroxidase shown by a 1.8 Å resolution crystallographic study at pH 4.5

“…The structure at pH 4.5 presented here may represent the physiological form of the resting enzyme and is the first example of a peroxidase with a typical pentacoordinated heine iron demonstrated by crystallography. This is consistent with the resonance Raman and NMR studies done on C. einereus peroxidase which showed that at a neutral pH the ferric state is characteristic of a fivecoordinated high-spin heme [26,27]. In CcP, LiP, MnP, and ascorbate peroxidase, a water molecule occupies the 6th position of each heme [14][15][16][17].…”

Kinetic Stability of Designed Glycosylation Mutants of Coprinus cinereus Peroxidase

“…The effects of the G156F and N157F mutations, however, were not additive and the double mutant G156F-N157F was more stable than each of the single mutants, indicating that the two phenylalanine side chains interact positively in the native state. NMR analyses of the single mutants and the double mutant support this finding (19). The equivalent residues of HRP C, F142 and F143, also interact, as seen in the crystal structure (28).…”

Binding of Iodide to Arthromyces ramosus Peroxidase Investigated with X-ray Crystallographic Analysis, 1H and 127I NMR Spectroscopy, and Steady-state Kinetics

“…The hyperfine-shifted spectrum in the region of 60 -90 ppm is characterized by three peaks (a, b, and c), the size of the last peak being about twice of that of peaks a and b. The spectral pattern is similar to that of C. cinereus peroxidase whose amino acid sequence is almost identical to that of ARP (28,40,41). Therefore, it is most likely that peaks a, b, and c arise from protons of heme peripheral methyls at 3, 8, and 1 plus 5, respectively.…”