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...
We describe cDNAs for a HSP90 homologue from Catharanthus roseus and studies on the regulation of expression. The largest cDNA (2670 bp) coded for a protein of 817 amino acids with a calculated size of 93,491 Da and a pI of 4.61. It contained a eucaryotic secretory signal, the endoplasmic reticulum (ER) targeting and retention signal (Lys-Asp-Glu-Leu), and the HSP90 protein family signature with one conservative exchange (Asn-Lys-Asp-Ile-Phe-Leu instead of Asn-Lys-Glu-Ile-Phe-Leu). RNA blots revealed a transcript of 2.8-2.9 kb, and genomic DNA blots suggested a single gene. The expression was analysed with antiserum against a fusion protein expressed in Escherichia coli. Immunoblots revealed a protein of 93 +/- 1.5 kDa (often a doublet) only in the membrane fraction, and sucrose density gradients suggested association with the ER. The protein was constitutively expressed in C. roseus cell cultures grown at 25 degrees C, and expression was apparently unaffected by various stress conditions, such as heat, high sucrose, elicitor from Phytophthora megasperma or yeast extract. It was not detectable in young C. roseus plants at room temperature, and heat shock for several hours at 37 degrees C was necessary to obtain detectable expression. In maize (Zea mays), a cross-reacting protein was detectable in cell cultures, but not in young plants. The results suggested that the cloned protein is not a major component in the heat shock response. We propose a chaperone role in the assembly and processing of cell wall components and other secreted proteins, i.e. functions that are very active in cells with a high rate of growth and division.
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