Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae.
An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.
Wang
Linsong
L
Risø National Laboratory, Plant Research Department, PRD-301, Plant Quality, DK-4000 Roskilde, Denmark.
Burhenne
Kim
K
Kristensen
Brian K
BK
Rasmussen
Søren K
SK
D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D031215:Armoracia / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D018517:Plant Roots / Q000201:enzymology / Q000378:metabolism; D031224:Raphanus / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000872:Anthocyanins; C066957:pelargonidin; D010544:Peroxidases