Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures.
Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation.
Schwarz
H
H
Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Braunschweig, Germany.
Liu
B
B
Peters
S
S
Barillas
W
W
Beerhues
L
L
D000085:Acetates / Q000378:metabolism; D000595:Amino Acid Sequence; D018929:Cell Culture Techniques; D029966:Centaurium / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D018745:Genome, Plant; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D008861:Microsomes; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016366:Open Reading Frames; D054883:Oxylipins / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000085:Acetates; D003517:Cyclopentanes; D018076:DNA, Complementary; D006023:Glycoproteins; D054883:Oxylipins; D010940:Plant Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C072239:methyl jasmonate; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase