Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

Menting, John G.; Scopes, Robert K.; Stevenson, Trevor W.

doi:10.1104/pp.106.2.633

Cited by 40 publications

(34 citation statements)

References 36 publications

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“…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).…”

Section: Sources and Structures Of Proanthocyanidinsmentioning

confidence: 99%

Proanthocyanidins – a final frontier in flavonoid research?

2004

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Summary Proanthocyanidins are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They are present in the fruits, bark, leaves and seeds of many plants, where they provide protection against predation. At the same time they give flavor and astringency to beverages such as wine, fruit juices and teas, and are increasingly recognized as having beneficial effects on human health. The presence of proanthocyanidins is also a major quality factor for forage crops. The past 2 years have seen important breakthroughs in our understanding of the biosynthesis of the building blocks of proanthocyanidins, the flavan‐3‐ols (+)‐catechin and (–)‐epicatechin. However, virtually nothing is known about the ways in which these units are assembled into the corresponding oligomers in vivo. Molecular genetic approaches are leading to an understanding of the regulatory genes that control proanthocyanidin biosynthesis, and this information, together with increased knowledge of the enzymes specific for the pathway, will facilitate the genetic engineering of plants for introduction of value‐added nutraceutical and forage quality traits. Contents Summary9I.9II.10III.12IV.13V.14VI.16VII.23VIII.242525

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Section: Sources and Structures Of Proanthocyanidinsmentioning

confidence: 99%

Proanthocyanidins – a final frontier in flavonoid research?

2004

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“…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.…”

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confidence: 99%

“…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).…”

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

Takahashi

Zhao

O’Maille

et al. 2005

Journal of Biological Chemistry

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The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio-and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1␤-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1␤(OH)EA versus 3␣(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified activesite residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1␤(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1␤(OH)EA, 2␤(OH)EA, and 3␤(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted ϳ1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6-and 5.7-fold increases in their K m values for the 1␤(OH)EA intermediate, respectively. A role for Ile 486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1␤(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher K m for EA and a 4.4-fold slower turnover rate (k cat ) for 1␤(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser 368 and Ile 486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.The mevalonate and methylerythritol phosphate pathways are responsible for the biosynthesis of isoprenoids, a diverse group of organic natural products found in animals, plants, fungi, insects, and bacteria. Isoprenoids are further divided into classes of primary and secondary metabolites. Isoprenoids that are primary metabolites include sterols, carotenoids, hormones, and long chain hydrocarbons used to tether particular enzymes to membrane systems, compounds essential for viability. Isoprenoids classified as secondary metabolites include monoterpenes, sesquiterpenes, diterpenes, and triterpenes, and many of these mediate interactions between organisms and their environments (1-5).Capsidiol is a bicyclic dihydroxylated sesquiterpene produced by several solanaceous plants in response to pathogen or elicitor challenge (6 -10) and is considered an important plant defense response because it can prevent the germination and growth of several fungal species (11). Capsidiol biosynthesis is regulated by the expression of two key enzymes (see Scheme 1...

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“…1). The enzyme is considered to be a member of the cytochrome P450 family from its biochemical characteristics such as association with microsomal membrane fraction, dependence on NADPH and O 2 , and sensitivity to inhibitors [1,2]. However, it has been di¤cult to purify and isolate the enzyme in su¤cient amounts to carry out further investigations, such as amino acid sequencing and cDNA isolation.…”

Section: Introductionmentioning

confidence: 99%

Expression of chimeric P450 genes encoding flavonoid‐3′,5′‐hydroxylase in transgenic tobacco and petunia plants¹

et al. 1999

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Flavonoid-3P P,5P P-hydroxylase (F3P P5P PH), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3P P,5P P-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3P P5P PH. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3P P,5P P-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3P P,5P P-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3P P5P PH activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3P P5P PH activity between tobacco and petunia is discussed.z 1999 Federation of European Biochemical Societies.

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Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

Cited by 40 publications

References 36 publications

Proanthocyanidins – a final frontier in flavonoid research?

Proanthocyanidins – a final frontier in flavonoid research?

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

Expression of chimeric P450 genes encoding flavonoid‐3′,5′‐hydroxylase in transgenic tobacco and petunia plants¹

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Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers

Cited by 40 publications

References 36 publications

Proanthocyanidins – a final frontier in flavonoid research?

Proanthocyanidins – a final frontier in flavonoid research?

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

Expression of chimeric P450 genes encoding flavonoid‐3′,5′‐hydroxylase in transgenic tobacco and petunia plants1

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Expression of chimeric P450 genes encoding flavonoid‐3′,5′‐hydroxylase in transgenic tobacco and petunia plants¹