Purification, cDNA cloning and functional expression of NADPH‐cytochrome P450 reductase from Centaurium erythraea cell cultures

Abstract

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.

NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

“…The treatments with MeJA and SA resulted in induction of WsCPR2 while the expression of WsCPR1 remained unchanged. These observations are in agreement with earlier report on C. erythrium where only CPR2 was induced on treatment with MeJA [22]. In A .…”

“…Nothapodytes foetida and Hybrid poplar ( Populus trichocarpa × Populus deltoides ) [17], [18] contain three paralogs while Helianthus tuberosus , Petroselinum crispum , Arabidopsis thaliana, Centaurium erythrae and Gossipium hirusitum have two CPR homologs. [19], [20], [21], [22], [23]. Similarly, single form of CPR has been characterised from some plants like Coleus blumei , Papaver somniferum , Taxus cuspidate , Vigna radiate [24], [25], [26], [27].…”

Class II Cytochrome P450 Reductase Governs the Biosynthesis of Alkaloids

“…CPR1s belonging to class I, such as ATR1, are constitutively expressed, while CPR2s from class II (e.g. ATR2) are inducible by environmental stimuli such as wounding, pathogen infection, or light exposure (Koopmann and Hahlbrock, 1997;Ro et al, 2002;Schwarz et al, 2009;Mizutani and Ohta, 2010;Yang et al, 2010, Rana et al, 2013. These observations suggest that each CPR isoform has dedicated physiological roles, ensuring that plants can meet the reductive demand of P450-mediated reactions.…”

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…In contrast to animals, which contain a single CPR (Porter et al, 1990), plants have multiple CPR isoforms that fall into two classes structurally distinguishable by their different N termini (Urban et al, 1997;Ro et al, 2002). In Arabidopsis and Centaurium erythraea, the expression of class II CPR is induced by wounding, light treatment, and elicitation, whereas class I CPR is constitutively regulated (Nisimoto, 1986;Meijer et al, 1993;Schwarz et al, 2009). Consistently, the transcript for rowan class II CPR, which contained all essential structural motifs, could be isolated from cell cultures after the addition of elicitor.…”