Purification and kinetic characterization of two peroxidases of Selaginella martensii Spring. involved in lignification.
Two cationic peroxidases from Selaginella martensii Spring. (SmaPrx2 and SmaPrx3) were purified using a three-step protocol which includes ammonium sulfate precipitation, adsorption chromatography on phenyl sepharose and cationic exchange chromatography on SP sepharose. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3 kDa and 45.6 kDa, respectively, according to MALDI-TOF/TOF. The isoelectric points were estimated in 9.2 and 9.5 for SmaPrx2 and SmaPrx3, respectively, according to isoelectrofocusing. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403 nm for SmaPrx2 and 404 nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the in vitro capacity of using sinapyl alcohol as substrate and the low K(m) in the μM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignins biosynthesis.
Department of Animal Biology, Plant Biology and Ecology, University of A Coruña, E-15071 A Coruña, Spain.
Teresa
T
Martínez-Cortés
Federico
F
Pomar
José Manuel
JM
Espiñeira
Fuencisla
F
Merino
Esther
E
Novo-Uzal
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008031:Lignin / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010666:Phenylpropionates / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020033:Protein Isoforms; D040901:Proteomics; D032503:Selaginellaceae / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D010666:Phenylpropionates; D010940:Plant Proteins; D020033:Protein Isoforms; C496130:sinapyl alcohol; D008031:Lignin; D010544:Peroxidases