Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.).
A cationic (pI 8.3) wall-bound peroxidase has been purified to homogeneity from suspension-cultured cells of French bean (Phaseolus vulgaris L.). The enzyme was a glycoprotein and its M(r) was 46,000 as determined by SDS/Page and h.p.l.c. gel filtration. It was localized biochemically to microsomes and the cell wall, and the latter subcellular distribution was confirmed by immunogold techniques. The native enzyme showed absorption maxima at 403, 500 and 640 nm, with an RZ (A405/A280) of 3.3. The peroxidase oxidized guaïacol and natural phenolic acids. By desorption-chemical-ionization mass spectrometry the enzyme was found to oxidize the model compound, ferulic acid, into dehydrodiferulic acid. Kinetics studies indicated an apparent Km of 113.3 +/- 22.9 microM and a Vmax of 144 mumol.min-1.nmol-1 of protein at an H2O2 concentration of 100 microM. In comparison with a second French-bean peroxidase (FBP) and horseradish peroxidase, as a model, it acted with a 6-10-fold higher specificity in this capacity. It is a member of the peroxidase superfamily of bacterial, fungal and plant haem proteins by virtue of its highly conserved amino acid sequence within the proximal and distal haem-binding sites. This is good evidence that this particular FBP may function in constructing covalent cross-linkages in the wall during development and response to pathogens.
Zimmerlin
A
A
Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Egham, Surrey, U.K.
Wojtaszek
P
P
Bolwell
G P
GP
D000595:Amino Acid Sequence; D001704:Biopolymers; D002473:Cell Wall / Q000201:enzymology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003373:Coumaric Acids / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D006865:Hydrogenation; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D010946:Plants, Medicinal; D016415:Sequence Alignment
D001704:Biopolymers; D003373:Coumaric Acids; D007527:Isoenzymes; C004999:ferulic acid; D010544:Peroxidases