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1.
A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3 of the aromatic rings of the ester 4-coumaroyl-4-hydroxyphenyllactate giving rise to rosmarinic acid.Abbreviations Caf-pHPL
caffeoyl-4-hydroxyphenyllactate
- DHPL
3,4-dihydroxyphenyllactic acid
- pC-DHPL
4-coumaryl-3,4-dihydroxyphenyllactate
- pC-pHPL
4-coumaryl-4-hydroxyphenyllactate
- pHPL
4-hydroxyphenyllactic acid
- RA
rosmarinic acid
The financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged. 相似文献
2.
Maike Petersen Elisabeth Häusler Juliane Meinhard Barbara Karwatzki Claudia Gertlowski 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):171-179
Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL
3,4-dihydroxyphenyllactate
- DHPP
3,4-dihydroxyphenylpyruvate
- pHPL
4-hydroxyphenyllactate
- pHPP
4-hydroxyphenylpyruvate
- RA
rosmarinic acid 相似文献
3.
Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase. 相似文献
4.
CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis 总被引:3,自引:0,他引:3
Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis. 相似文献
5.
Maike Petersen Elisabeth Szabo Juliane Meinhard Barbara Karwatzki Claudia Gertlowski Bettina Kempin Elisabeth Fuß 《Plant Cell, Tissue and Organ Culture》1995,43(2):89-92
This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH
cinnamic acid 4-hydroxylase
- 4CL
4-coumarate:CoA ligase
- HPPR
hydroxyphenylpyruvate reductase
- 3-H
hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase
- 3-H
hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase
- PAL
phenylalanine ammonia-lyase
- RAS
rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase)
- TAT
tyrosine aminotransferase 相似文献
6.
Maike Petersen Yana Abdullah Johannes Benner David Eberle Katja Gehlen Stephanie Hücherig Verena Janiak Kyung Hee Kim Marion Sander Corinna Weitzel Stefan Wolters 《Phytochemistry》2009,70(15-16):1663-1679
Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be “invented” for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration. 相似文献
7.
P. B. Danielson J. L. M. Foster M. M. McMahill M. K. Smith J. C. Fogleman 《Molecular & general genetics : MGG》1998,259(1):54-59
In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects
members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds.
A degenerate 3′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that
members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships.
Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those
of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs.
Received: 14 August 1997 / Accepted: 24 March 1998 相似文献
8.
9.
Y. V. Inyushkina K. V. Kiselev V. P. Bulgakov Yu. N. Zhuravlev 《Biochemistry. Biokhimii?a》2009,74(8):917-924
10.
Cytochromes P450 in phenolic metabolism 总被引:2,自引:0,他引:2
Jürgen Ehlting Björn Hamberger Rachel Million-Rousseau Danièle Werck-Reichhart 《Phytochemistry Reviews》2006,5(2-3):239-270
Three independent cytochrome P450 enzyme families catalyze the three rate-limiting hydroxylation steps in the phenylpropanoid pathway leading to the biosynthesis of lignin and numerous other phenolic compounds in plants. Their characterization at the molecular and enzymatic level has revealed an unexpected complexity of phenolic metabolism as the major route involves shikimate/quinate esters and alcohol/aldehyde intermediates. Engineering expression of CYP73s (encoding cinnamate 4-hydroxylase), CYP98s (encoding 4-coumaroylshikimate 3′-hydroxylase) or CYP84s (encoding coniferaldehyde 5-hydroxylase) leads to modified lignin and seed phenolic composition. In particular CYP73s and CYP98s also play essential roles in plant growth and development, while CYP84 constitutes a check-point for the synthesis of syringyl lignin and sinapate esters. Although recent data shed new light on the main path for lignin synthesis, they also raised new questions. Mutants and engineered plants revealed the existence of (an) alternative pathway(s), which most likely involve(s) different precursors and oxygenases. On the other hand, phylogenetic analysis of plant genomes show the existence of P450 gene duplications in each family, which may have led to the acquisition of novel or additional physiological functions in planta. In addition to the main lignin pathway, P450s contribute to the biosynthesis of many bioactive phenolic derivatives, with potential applications in medicine and plant defense, including lignans, phenylethanoids, benzoic acids, xanthones or quinoid compounds. A very small proportion of these P450s have been characterized so far, and rarely at a molecular level. The possible involvement of P450s in salicylic acid is discussed. 相似文献
11.
The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root
clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for
growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected
by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones,
while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones. 相似文献
12.
Ojima K Breitenbach J Visser H Setoguchi Y Tabata K Hoshino T van den Berg J Sandmann G 《Molecular genetics and genomics : MGG》2006,275(2):148-158
A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a β-carotene accumulating mutant. It consists of 3,166 bp and contains 17
introns. For the β-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The
resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this β-carotene oxygenase encodes a cytochrome
P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an
oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by
genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from β-carotene to astaxanthin formation
by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of β-carotene followed by 3-hydroxylation. A
hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both
β-ionone ends. 相似文献
13.
M. Teresa Donato José V. Castell M. José Gómez-Lechón 《In vitro cellular & developmental biology. Animal》1994,30(12):825-832
Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat
hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase
activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid
loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable
in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects
of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the
best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different
induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and
a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in
P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation). 相似文献
14.
Bedia Palabiyik Semian Karaer Nazli Arda Sidika Erturk Toker Guler Temizkan Steven Kelly Aysegul Topal Sarikaya 《Biologia》2008,63(3):450-454
Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one
of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs
and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL)
containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence
of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The
enzyme activity was confirmed by HPLC analysis, using testosterone as substrate. 相似文献
15.
Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries.
Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic
conditions. This study shows that this O2- and NADPH/FMNH2-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain.
The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype.
The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the
rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K
d ≈ 65 μM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP. 相似文献
16.
We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol
profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14
proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce
stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K
m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported
with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal
level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together
with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway
in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
18.
Morant M Schoch GA Ullmann P Ertunç T Little D Olsen CE Petersen M Negrel J Werck-Reichhart D 《Plant molecular biology》2007,63(1):1-19
A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . 相似文献
19.
Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed
to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol
is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes
CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and
are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation
of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of
free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher
levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings
of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore,
our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols. 相似文献
20.
Bixia Zhang Gerhard R. Munske Vitaliy I. Timokhin John Ralph Dmitri R. Davydov Wilfred Vermerris Scott E. Sattler ChulHee Kang 《The Journal of biological chemistry》2022,298(4)
Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3′-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3′H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide–binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering. 相似文献