Characterization of basic p-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming Picea abies suspension culture.
A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K(m) values in microM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V(max)/K(m)) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.
Koutaniemi
Sanna
S
Department of Applied Biology, University of Helsinki, Finland. sanna.koutaniemi@helsinki.fi
Toikka
Merja M
MM
Kärkönen
Anna
A
Mustonen
Maaret
M
Lundell
Taina
T
Simola
Liisa K
LK
Kilpeläinen
Ilkka A
IA
Teeri
Teemu H
TH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D028222:Picea / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D046509:Tissue Culture Techniques / Q000379:methods
D003373:Coumaric Acids; D018076:DNA, Complementary; D007527:Isoenzymes; D010636:Phenols; D010940:Plant Proteins; D008031:Lignin; C010559:coniferyl alcohol; D010544:Peroxidases