Biochemical characterization of the major sorghum grain peroxidase.
The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.
Dicko
Mamoudou H
MH
Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands. mdicko@univ-ouaga.bf
Gruppen
Harry
H
Hilhorst
Riet
R
Voragen
Alphons G J
AG
van Berkel
Willem J H
WJ
D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000737:chemistry* / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D045868:Sorghum / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity
D002241:Carbohydrates; D010940:Plant Proteins; D009195:Peroxidase