Molecular and biochemical investigations on hydroxycinnamoyltransferases from Coleus blumei and Glechoma hederacea Lignin opus:2907 ths Prof. Dr. Petersen Maike Petersen, Maike (Prof. Dr.) 2010-05-19 Chlorogensäure Philipps-Universität Marburg Coleus Hydroxycinnamic acid esters are prominent natural products in Lamiaceae. Besides hydroxycinnamoylshikimate and -quinate, which are rather ubiquitous in the plant kingdom, rosmarinic acid is found predominantly in Boraginaceae and in the subfamily Nepetoideae of the Lamiaceae. The biosynthetic pathways towards monolignols, chlorogenic acid and rosmarinic acid are extensively investigated. The hydroxycinnamoyltransferases (HCTs), involved in the biosynthesis of these esters, belong to the superfamily of BAHD acyltransferases and accept hydroxycinnamoyl-CoAs as hydroxycinnamoyl donors. They transfer hydroxycinnamic acids from CoA to different acceptor substrates (HST: shikimate, HQT: quinate, RAS hydroxyphenyllactates) and show generally an explicit substrate specificity. The first cDNA encoding a hydroxycinnamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS) was cloned from Coleus blumei. RAS enzyme activity is essential for the formation of rosmarinic acid. In this work the cDNA encoding a hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (CbHST) could be isolated from the same plant species. The proteins were heterologously expressed in E. coli and the substrate acceptance of both enzymes were tested. Interestingly, besides ester formation also amide formation can be catalyzed. CbHST transfers CoA-activated cinnamic acids (p-coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, sinapoyl-CoA) to shikimate but not to quinate or acceptor substrates utilized by CbRAS. Also 3-hydroxyanthranilate, 2,3-dihydroxybenzoate, 3-hydroxybenzoate and 3-aminobenzoate yielded products. CbRAS transfers the same CoA-activated cinnamic acids (except sinapoyl-CoA) to D-(hydroxy-)phenyllactic acids (D-4-hydroxyphenyllactate, D-3,4-dihydroxyphenyllactate, D-phenyllactate), but not to quinate or shikimate. In addition, the D-amino acids D-phenylalanine, D-tyrosine and D-DOPA are accepted as acceptor substrates by CbRAS. The Km-values for a specific substrate were dependent on the second substrate used in the assay. This is an indication of the plasticity of the active sites (induced fit). The coexistence of both enzymes in one species, the high substrate specificity and the kinetic parameters indicate, that CbHST is a specific enzyme of monolignol biosynthesis and CbRAS a specific enzyme of rosmarinic acid biosynthesis. p-coumaroyl-CoA is the preferred substrate. To identify important amino acid residues, CbRAS mutants H152A, D156A, D377A, R285A, W380L and L136P were generated. CbRAS mutants showed a residual activity +amp;amp;lt; 1 % in comparison to the wild type RAS. An involvement of H152 in catalysis is anticipated. To perform crystallization experiments, two different purification procedures were established, which allow the purification of CbRAS to apparent homogeneity. Glechoma hederacea accumulates rosmarinic acid and chlorogenic acid in parallel. Therefore Glechoma is suitable for the detailed investigation of the three biosynthetic pathways. By sequence alignment of known RAS, HST and HQT degenerated primers were designed. The 5´- and 3´-ends were amplified with help of RACE-PCR and enabled the deduction of 6 full-length sequences. All translated protein sequences exhibit the conserved sequence motivs HxxxD and DFGWG. Three enzymes could be expressed functionally in E. coli: GhRAS-l is a hydroxycinamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyltransferase and transfers p-coumaric acid and caffeic acid from CoA to p-hydroxyphenyllactate (pHPL) and 3,4-dihydroxyphenyllactate (DHPL), but not to shikimate or quinate. GhHST-k und GhHST-l transfer both CoA-activated acids to shikimate, but not to pHPL, DHPL or quinate. GhRAS-k has an identity of 91 % to GhRAS-l, but showed no enzyme activity. A pseudogen is supposed. On the basis of the amino acid sequence, the putative GhHQT-k and GhHQT-l have the highest similarity to a HQT from Cynara cardunculus (50 % and 45 %). No enzyme activity could be detected. Possibly GhHQT-k and GhHQT-l are no HQTs, but HCTs with unknown substrate specificity so far. A phylogenetic tree of the isolated RAS-, HST- and putative HQT- sequences from Coleus and Glechoma together with other biochemical characterized BAHD-acyltransferases showes that the HCTs are closely related. The Phylogenie indicates, that RAS, HST and HQT evolved possibly by duplication and diversification. Rosmarinic acid application/pdf 2010 https://doi.org/10.17192/z2010.0168 Fachbereich Pharmazie BAHD-acyltransferase Hydroxyzimtsäureester sind bedeutende sekundäre Inhaltsstoffe der Lamiaceae. Neben Hydroxycinnamoylshikimat und -chinat, die im Pflanzenreich nahezu ubiquitär verbreitet sind, ist Rosmarinsäure hauptsächlich in den Boraginaceae und der Unterfamilie Nepetoideae der Lamiaceae zu finden. Die Biosynthesewege dieser Phenylpropanderivate sind aufgrund weitgehend verstanden. Die Hydroxycinnamoyltransferasen (HCTs), die an der Biosynthese dieser Ester beteiligt sind, gehören zur Superfamilie der BAHD-Acyltransferasen und übertragen Hydroxyzimtsäurereste von Hydroxycinnamoyl-CoA-Derivaten auf verschiedene Akzeptoren (HST: Shikimat, HQT: Chinat, RAS: Hydroxyphenyllactat). Dabei sind sie in der Regel sehr spezifisch für ihre Substrate. Mit der Klonierung einer cDNA aus Coleus blumei, die für eine Hydroxycinnamoyl-CoA: Hydroxyphenyllactat Hydroxycinnamoyltransferase (CbRAS) codiert, gelang es Berger et al. (2006) erstmals, das Gen des charakteristischen Enzyms der Rosmarinsäurebiosynthese zu isolieren. Aus derselben Pflanzenart konnte in dieser Dissertation die cDNA, die für eine Hydroxycinnamoyl-CoA:Shikimat Hydroxycinnamoyltransferase (CbHST) codiert, isoliert werden. Beide Proteine wurden in E. coli heterolog exprimiert und die Substratspezifitäten untersucht. Beide HCTs besitzen eine breitere Substratspezifiät als erwartet und können neben Estern auch Amide bilden. Die CbHST überträgt CoA-aktivierte Zimtsäuren (p-Cumaroyl-CoA, Caffeoyl-CoA, Cinnamoyl-CoA, Feruloyl-CoA und Sinapoyl-CoA) auf Shikimat, nicht aber auf Chinat oder die Akzeptorsubstrate der CbRAS. Mit 3-Hydroxyanthranilsäure, 2,3-Dihydroxybenzoesäure, 3-Hydroxybenzoesäure und 3-Aminobenzoesäure wurde ebenfalls eine Produktbildung beobachtet. Die CbRAS überträgt dieselben CoA-aktivierten Zimtsäuren (außer Sinapoyl-CoA) auf D-p-Hydroxyphenyllactat (pHPL), D-Dihydroxyphenyllactat (DHPL) und D-Phenyllactat, nicht aber auf Chinat oder Shikimat. Die D-Aminosäuren D-Phenylalanin, D-Tyrosin und D-DOPA sind ebenfalls Substrate der CbRAS. Die Km-Werte für ein spezifisches Substrat waren immer abhängig von dem zweiten, konstant gehaltenen Substrat. Dies ist ein Hinweis für die Plastizität der aktiven Zentren (induced fit). Die Koexistenz beider Enzyme in einer Pflanzenart, die ausgeprägte Substratspezifität und der Vergleich der enzymkinetischen Parameter führt zu dem Ergebnis, dass es sich bei der CbHST und der CbRAS um spezifische Enzyme der Monolignol- (CbHST) bzw. der Rosmarinsäuresynthese (CbRAS) handelt, deren bevorzugtes Donorsubstrat p-Cumaroyl-CoA ist. Zur Identifizierung von funktionell und/oder strukturell wichtigen Aminosäuren wurden gezielt die CbRAS-Mutanten H152A, D156A, D377A, R285A, W380L und L136P generiert. Die Einzelmutationen in diesen konservierten Bereichen führten zu einer drastischen Reduktion der Enzymaktivität (Restaktivität unter 1%). Vermutlich ist das H152 des 152HxxxD156-Motivs die katalytische Base. Es wurden zwei verschiedene Aufreinigungsverfahren entwickelt, mit deren Hilfe die CbRAS im zweistelligen mg-Bereich bis zur apparenten Homogenität aufgereinigt werden konnte. In ersten Kristallisationsversuchen wuchsen erste kleine Kristalle, bei denen es sich wahrscheinlich um CbRAS-Proteinkristalle handelt. Glechoma hederacea produziert Rosmarinsäure und Chlorogensäure nebeneinander. Daher ist sie für die Untersuchung aller drei Biosynthesewege geeignet. Durch Nukleotidsequenzvergleich von bekannten RAS, HST und HQT wurden degenerierte Primer abgeleitet, die unter Verwendung der RACE-PCR-Technik zur selketiven Amplifikation von sechs cDNA-Klonen führten. Die translatierten Proteinsequenzen weisen die für BAHD-Acyltransferasen typischen konservierten Motive HxxxD und DFGWG auf. Drei Enzyme konnten funktionell in E. coli exprimiert werden: GhRAS-l ist eine Hydroxycinnamoyl-CoA:Hydroxyphenyllactat Hydroxycinnamoyltransferase, die spezifisch p-Cumaroyl-CoA und Caffeoyl-CoA auf pHPL und DHPL, nicht aber auf Shikimat oder Chinat überträgt. Die GhHST-k und die GhHST-l übertragen die beiden CoA-aktivierten Säuren selektiv auf Shikimat, nicht aber auf pHPL, DHPL oder Chinat. Die GhRAS-k mit 91% Sequenzidentität zur aktiven GhRAS-l war in Enzymtests nicht aktiv. Ein Pseudogen wird vermutet. Die putative GhHQT-k und die putative GhHQT-l weisen die höchste Aminosequenzähnlichkeit (50 % und 45 %) zur HQT aus Cynara cardunculus auf. Sie waren in Enzymtests jedoch nicht aktiv. Daher bleibt fraglich, ob es sich um HQTs oder um Enzyme mit bislang unbekannter Enzymaktivität handelt. Die Verwandtschaftsverhältnisse der isolierten RAS-, HST- und putativen HQT-Sequenzen aus Coleus und Glechoma zu bekannten BAHD-Acyltransferasen zeigt die enge evolutionäre Beziehung der HCTs zueinander und gibt einen Hinweis, dass RAS, HST und HQT möglicherweise durch Genverdopplung und Diversifikation auseinander hervorgegangen sind. doctoralThesis Rosmarinsäure Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg ppn:22570725X Lamiaceae Hydroxycinnamoyltransferase Pharmazeutische Biologie Molekularbiologische und biochemische Untersuchungen von Hydroxycinnamoyltransferasen aus Coleus blumei und Glechoma hederacea Glechoma German BAHD-Acyltransferase 2011-08-10 Lignin Chlorogenic acid Botanical sciences Pflanzen (Botanik) 2010-07-08 Sander, Marion Sander Marion urn:nbn:de:hebis:04-z2010-01685 monograph Petersen M, Häusler E, Karwatzki B, Meinhard J (1994). The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei. Plant Cell Tiss. 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Plant Physiol 130:466-476 Stöckigt J, Zenk MH (1975) Chemical syntheses and properties of hydroxycinnamoyl‐coenzyme A derivates. Z Naturforsch 30c:352-358 Sondheimer E (1964) Chlorogenic acid and related depsides. Bot Rev 30:667-712 Lepepelley M, Cheminade G, Tremillon N, Simkin A, Caillet V, McCarthy J (2007) Chlorogenic acid synthesis in coffee: An analysis of CGA content and real-time RT-PCR expression of HCT, HQT, C3H1, and CCoAOMT1 genes during grain development in C. canephora. Plant Sci. 172:978-996 hol acyltransferase (MdAAT2) from Apple (cv. Golden Delicious). Phytochemistry 67:658-667 Peng XX, Bai GB, Li WD (submitted 2009). Cloning and characterization of a cDNA coding a hydroxycinnamoyl transferase involved in chlorogenic acid biosynthesis in Lonicera japonica. Xia Y, Nikolau BJ, Schnable PS (1996) Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax acuumulation 8:1291-1304 CoA:quinate hydroxycinnamoyl transferase from higher plants. Phytochemistry 18:929- Rogers R (2008) Coleus: rainbow foliage for containers and gardens. Timber Press, Inc. Petersen M (2007) Current status of metabolic phytochemistry. Phytochemistry 68:2847- 2860 Petersen M (1997) Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus. Phytochemistry 45:1165-1172 DAC (2009) Deutscher Arzneimittel-Codex, Deutscher Apothekerverlag Stuttgart D'Auria JC (2006) Acyltransferases in plants: a good time to be BAHD. Curr Opin Plant Biol 9:331-340 Acyltransferasen erstellt. Die gefundenen Verwandtschaftsverhältnisse sind mit den von D´Auria (2006) beschriebenen Ergebnissen vergleichbar. Daher wurde die damals eingeführte Nummerierung der Klassen beibehalten. Als Basis für den Stammbaum dienten Proteinsequenzen aus der UniProt-Datenbank, deren Substratspezifität bekannt ist. Abkürzungen Klasse I: At3AT1 Arabidopsis thaliana Cumaroyl-CoA:Cyanidin-3-O-glucose Hydroxycinnamoyltransferase; At3AT2 Arabidopsis thaliana Cumaroyl-CoA:Cyanidin-3-O-glucose Hydroxycinnamoyltransferase; MtMAT3 Medicago truncatula Isoflavon-7-O-glykosid Malonyltransferase; MtMAT1 Medicago truncatula Isoflavon-7-O-glykosid Malonyltransferase; At5MaT Arabidopsis thaliana Malonyl- CoA:Anthocyan-5-O-glucosid 6´´-O-Malonyltransferase; Dm3MAT2 Dendranthema x morifolium Anthocyanidin-3- O-glucosid 3´´,6´´-O-Dimalonyltransferase; Dm3MAT1 Dendranthema x morifolium Anthocyanidin-3-O-glucosid 6´´-O-Malonyltransferase; Sc3MaT Senecia curentus Malonyl-CoA:Anthocyanidin 3-O-glucosid 6´´-O- Malonyltransferase; Dv3MAT Dhalia variabilis Malonyl-CoA:Anthocyanidin-3-O-glucosid 6´´-O- Malonyltransferase; Dv3MAT2 Dahlia variabilis Malonyl-CoA:Anthocyanidin-3-O-glucosid 6´´-O- Malonyltransferase; Dm3MAT3 Dendranthemum x morifolium Anthocyanidin-3-O-glucosid 6´´-O- Malonyltransferase; Pf3AT Perilla frutescens Hydroxycinnamoyl-CoA:Anthocyanin-3-O-glucosid 6´´-O- Hydroxycinnamoyl-transferase; Ss3MAT Salvia splendens Anthocyanin-3-O-glucosid 6´´-O- Hydroxycinnamoyltransferase; Pf5MaT Perilla frutescens Anthocyanin-5-O-glucosid 6´´-O-Malonyltransferase; Ss5MaT Salvia splendens Malonyl-CoA:Anthocyanin-5-O-Glucosid 6´´´-O-Malonyltransferase; Gt5AT Gentiana triflora Hydroxycinnamoyl-CoA:Anthocyanin-5-O-glucosid 6´´´-O-Acyl-transferase; NtMAT1 Nicotiana tabacum Malonyl-CoA:Flavonoid/Naphtholglucosid Acyltransferase; Vh3MAT2 Verbena hybrida Quercetin-3-O-glucosid 6´´-O-Malonyltransferase; Vh3MAT1 Verbena hybrida Flavonol-3-O-glucosid 6´´-O-Malonyltransferase; Lp3MAT1 Lamium purpureum Flavonol-3-O-glucosid 6´´-O-Malonyltransferase; Klasse II: -Klasse III: CbBEAT Clarkia breweri Acetyl-CoA:Benzylalkohol Acetyltransferase; CcBEAT1-3 Clarkia concinna Acetyl-CoA:Benzylalkohol Acetyltransferasen; PsSalAT Papaver somniferum Salutaridinol 7-O-Acetyltransferase; CmAAT4 Cucumis melo Alkohol Acyltransferase; RhAAT1 Rosa hybrida Acetyl-CoA:Geraniol Acetyltransferase; SAAT Strawberry Alkohol Acyltransferase; VAAT Fragaria vesca Alkohol Acyltransferase; DAT Deacetylvindolin 4-O-Acetyltransferase; MAT Minovincinin 19-O-Acetyltransferase; RsVS Rauwolfia serpentina Vinorin-synthase; Ss5MaT2 Salvia splendens Malonyl-CoA:Anthocyanin-5-O-glucosid 6´´´-O-Malonyltransferase; Klasse IV: ACT Agmatin Cumaroyltransferase; Klasse Va: AtHHT1 Arabidopsis thaliana Hydroxycinnamoyl-CoA:ω-Hydroxysäure Hydroxycinnamoyl-transferase; DBBT Benzoyl-CoA:Taxan 2α-O-Benzoyltransferase; TAT Taxa-4(20),11(12)-dien- 5α-ol O-Acetyltransferase; DBAT 10-Deacetylbaccatin-III 10-O-Acetyltransferase; BAPT Baccatin III O- Phenylpropanoyltransferase; DBNTBT N-Debenzoyl-2´-deoxy-paclitaxel:N-Benzoyltransferase; AtSDT Arabidopsis thaliana Spermidin Disinapoyltransferase; AtSCT Arabidopsis thaliana Spermidin Dicumaroyltransferase; BanAAT Banana Alkohol Acyltransferase; CmAAT1 Cucumis melo Alkohol Acyltransferase; HMT/HLT Tigloyl-CoA:(-)-13α- hydroxymultiflorin/(+)-13α-hydroxylupanin O-Tigloyltransferase; CHAT (Z)-3-Hexen-1ol-O-Acetyltransferase; MpAAT1 Malus pumila Alkohol Acyltransferase; MdAAT2 Malus domestica Alkohol Acyltransferase; AMAT Anthraniloyl-CoA:Methanol Anthraniloyltransferase; CbBEBT Clarkia breweri Benzoyl-CoA:Benzylalkohol Benzoyltransferase; CmAAT3 Cucumis melo Alkohol Acyltransferase; NtBEBT Nicotiana tabacum Benzoyl- CoA:Benzylalkohol Benzoyltransferase; BPBT Benzoyl-CoA:benzylalkohol/phenylethalon Benzoyltransferase; Klasse Vb: CcHQT Cynara cardunculus Hydroxycinnamoyl-CoA:Chinat Hydroxycinnamoyltransferase; AtHCT Arabidopsis thaliana Hydroxycinnamoyl-CoA:Shikimat/Chinat Hydroxy-cinnamoyltransferase; TpHCT1 Trifolium pratense Hydroxycinnamoyl-CoA:Shikimat/Chinat Hydroxycinnamoyltransferase; TpHCT2 Trifolium pratense HydroxycinnamoylCoA:Maleinsäure Hydroxycinnamoyltransferase; NtHST Nicotiana tabacum Hydroxycinnamoyl- CoA:Shikimat Hydroxycinnamoyltransferase; CcHCT Coffea canephora Hydroxycinnamoyl-CoA:Shikimat/ Chinat Hydroxycinnamoyltransferase; CbHST Coleus blumei Hydroxycinnamoyl-CoA:Shikimat Hydroxycinnamoyltransferase; GhHST-k Glechoma hederacea Hydroxycinnamoyl-CoA:Shikimat Hydroxycinnamoyltransferase; GhHST-l Glechoma hederacea Hydroxycinnamoyl-CoA:Shikimat Hydroxycinnamoyltransferase; AsHHT1Avena sativa Hydroxycinnamoyl-CoA:Hydroxy-anthranilat N- Hydroxycinnamoyltransferase; NtHQT Nicotiana tabacum Hydroxycinnamoyl-CoA:Chinat Hydroxycinnamoyl- transferase; CsHQT1 Cynara scolymus Hydroxycinnamoyl-CoA:Chinat Hydroxycinnamoyltransferase; LjHQT Lonicera japonica Hydroxycinnamoyl-CoA:Chinat Hydroxycinnamoyltransferase; GhHQT-k Glechoma hederacea Hydroxycinnamoyltransferase ?; GhHQT-l Glechoma hederacea Hydroxycinnamoyltransferase?; CbRAS Coleus blumei Rosmarinsäuresynthase (Hydroxycinnamoyl-CoA-Hydroxyphenyllactat Hydroxycinnamoyltransferase); LaRAS Lavandula angustifolia Rosmarinsäuresynthase; MoRAS Melissa officinalis Rosmarinsäuresynthase; GhRAS-l Glechoma hederacea Rosmarinsäuresynthase; GhRAS-k Glechoma hederacea Rosmarin-säuresynthase Pseudogen?; HCBT Hydroxycinnamoyl/Benzoyl-CoA:Anthranilat N-Hydroxycinnamoyltransferase; AtSHT Arabidopsis thaliana Feruloyl-CoA:Spermidin Hydroxycinnamoyltransferase; Klasse VI: CFAT Coniferylalkohol Acetyltransferase Klasse VII: FsTRI10 Fusarium sporotichioides Trichothecen 3-O-Acetyltransferase; FgTRI101Fusarium graminearum Trichothecen 3-O-Acetyltransferase; Niggeweg R, Michael A, Martin C (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. 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