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

Abstract

SummaryWe investigated the P450 dependent¯avonoid 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¯avonoid 3¢,5¢-hydroxylase, but 3¢-hydroxylated products were also detected. The substrate speci®city 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¯avanones as well as¯avones, dihydro¯avonols and¯avonols, 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 avanone 3b-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¯ower buds from C. roseus identi®ed CHS expression preferentially in the epidermis, while F3¢5¢H was only detected in the phloem. The cell-type speci®c expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different¯avonoids.

Proanthocyanidins – a final frontier in flavonoid research?

“…These stereochemical differences are of major importance in proanthocyanidin biosynthesis, as all chiral intermediates in the flavonoid pathway up to and including leucoanthocyanidin are apparently of the 2,3-trans stereochemistry, raising important questions about the origin of the 2,3-cis stereochemistry of (-)-epicatechin, the commonest extension unit in proanthocyanidins (Foo & Porter, 1980). The B-ring hydroxylation pattern of the catechin/ epicatechin pair is determined by the presence or absence of the enzymes flavonoid 3 ′ -hydroxylase and flavonoid 3 ′ ,5 ′ -hydroxylase, cytochrome P 450 monooxygenases that act early in the pathway after formation of the flavanone naringenin (Menting et al ., 1994;Kaltenbach et al ., 1999;Schoenbohm et al ., 2000) (Fig. 2).…”

The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synthesis in Developing Grape Berries

“…1). Among other enzymes that use the same substrate are flavonoid 3#-hydroxylase and flavonoid 3#,5#-hydroxylase, which mediate the addition of hydroxyl groups to the B-ring of flavanones, flavones, and dihydroflavonols (Hagmann et al, 1983;Kaltenbach et al, 1999), and dihydroflavonol 4-reductase (DFR), which directly competes with FLS1 for the same substrate, dihydrokaempferol (Martens et al, 2002). VvFLS1 is expressed in leaves, tendrils, buds, inflorescences, and developing grape berries.…”

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

“…glucoside dhurrin (27), flavonoid 3Ј,5Ј-hydroxylase in anthocyanin biosynthesis (28,29), and several of the enzymes in the gibberellin biosynthetic pathway (30 -33). For example, flavonoid 3Ј,5Ј-hydroxylase (28,29) catalyzes the planar insertion of hydroxyl groups into the equivalent meta-positions (C-3Ј and C-5Ј) of the flavonoid B (phenolic) ring and yields 3Ј-, 5Ј-, and 3Ј,5Ј-hydroxylated reaction products (29).…”

“…For example, flavonoid 3Ј,5Ј-hydroxylase (28,29) catalyzes the planar insertion of hydroxyl groups into the equivalent meta-positions (C-3Ј and C-5Ј) of the flavonoid B (phenolic) ring and yields 3Ј-, 5Ј-, and 3Ј,5Ј-hydroxylated reaction products (29). ent-Kaurene oxidase (32) catalyzes the successive oxidation of the methyl substituent of ent-kaurene at C-19 en route to ent-kaurenoic acid.…”