Dokument

Zusammenfassung:
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.

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44. 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.
45. 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; 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