Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.)

Abstract

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCrosl, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia cofi recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 ag/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE1OH) and nerol-10-hydroxylase (NE1OH). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE1OH is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE1OH failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. P-4502 are the terminal oxidases of a large number of biotransformations. The enzymic reactions include metabo-1 This work was supported by the Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (SFB206).2Abbreviations: P-450, cytochrome(s) P-450; CA4H, cinnamicacid-4-hydroxylase; 14DM, lanosterol 14a-demethylase; FL3'H, flavonoid-3'-hydroxylase; FL3'5'H, flavonoid-3',5'-hydroxylase; GE1OH, geraniol-10-hydroxylase; LAH, lauric-acid-hydroxylase (in chain); NE1OH, nerol-10-hydroxylase; EROD, 7-ethoxyresorufin-0-deethylase; MX medium, growth medium; IM2 medium, alkaloid induction medium.lism of steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, pheromones, drugs, plant metabolites, and numerous other substances, including mutagens (19). The importance of these enzymes led in the last 10 years to a dramatic increase of molecular research in animal systems (20).The importance of P-450 enzymes in plants has been recognized (5), but the molecular analysis is lagging. The approach from purified protein to molecular biology has been difficult. In plants, the concentrations of these proteins are usually lower than in animals, the activities are unstable, the reconstitution of solubilized components to enzymically active complexes is difficult, and antisera against purified plant proteins or against P-450 enzymes from other sources most often recognize several...

Cinnamate 4‐hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli

“…Don, line CP3a), its maintenance on MX growth medium with subcultures every week, and the nutritional down-shift by transfer into a 8% sucrose solution have been described [20]. The PMG elicitor was a preparation from the cell walls of Phytophthora megasperma f. sp.…”

“…glyeinea [21]; it was used at a concentration of 1.2 mg/40 ml culture. The isolation of the membrane fractions has been published [20,22].…”

“…cDNA libraries. Poly(A)-rich RNA was isolated from C. roseus cell cultures induced for 7.5 h by a change from MX medium to 8% sucrose [20]. The construction of the libraries and the screening techniques have been described [30].…”

“…The separation of proteins for immunoblots was carried out in gels containing 0.1% sodium dodecyl sulfate and 8.5% polyacrylamide. The immunoreactions were performed with a secondary antibody coupled to alkaline phosphatase (Sigma Biochemicals) after protein transfer to nitrocellulose sheets [20].…”

“…The activities of C4H (with taC-labelled cinnamic acid) and of the Pas0-RED (cytochrome c reduction) were determined as described [20].…”