Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants

• Published in: The Plant journal : for cell and molecular biology Vol. 19; no. 2; pp. 183 - 193
• Main Authors: Kaltenbach, M, Schroder, G, Schmelzer, E, Lutz, V, Schroder, J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.07.1999; Blackwell Science
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Sequence; amino acid sequences; Base Sequence; Biological and medical sciences; Blotting, Southern; Catharanthus roseus; Cells, Cultured; chemical constituents of plants; complementary DNA; cytochrome P-450; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; dark; DNA, Complementary - chemistry; DNA, Complementary - genetics; enzyme activity; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; genbank/aj011862; genbank/aj131813; gene expression; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; histochemistry; Immunohistochemistry; lighting; Metabolism; Metabolism. Physicochemical requirements; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; nucleotide sequences; oxidoreductases; oxygenases; Petunia hybrida; Plant Cells; Plant physiology and development; Plants - enzymology; Plants - genetics; recombinant proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; substrates; Tissue Distribution; Immunohistochemistry; Heterologous system; Petunia hybrida; Enzyme; Tissue specificity; Homology; Catharanthus roseus; Gene expression; Flavonoid; Medicinal plant; Apocynaceae; Enzymatic activity; Complementary DNA; Flavonoid 3'-monooxygenase; Dicotyledones; Substrate specificity; Angiospermae; Spermatophyta; Oxidoreductases; Solanaceae; Kinetics; Localization; Secondary metabolism
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.1999.00524.x

We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3',5'-hydroxylase, but 3'-hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3'- as well as 3'5'-hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxylase and flavanone 3beta-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5'H substrates. Immunohistochemical localization of F3'5'H had not been performed before. A comparison of CHS and F3'5'H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3'5'H was only detected in the phloem. The cell-type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.