title: In vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures.
abstract: The anthranoid skeleton is believed to be formed by octaketide synthase (OKS), a member of the type III polyketide synthase (PKS) superfamily. Recombinant OKSs catalyze stepwise condensation of eight acetyl units to form a linear octaketide intermediate which, however, is incorrectly folded and cyclized to give the shunt products SEK4 and SEK4b. Here we report in vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures. Unlike field- and in vitro-grown shoots which accumulate anthraquinones, cell cultures mainly contained tetrahydroanthracenes, formation of which was increased 2.5-fold by the addition of yeast extract. The elicitor-stimulated accumulation of tetrahydroanthracenes was preceded by an approx. 35-fold increase in OKS activity. Incubation of cell-free extracts from yeast-extract-treated cell cultures with acetyl-CoA and [2-(14)C]malonyl-CoA led to formation of torosachrysone (tetrahydroanthracene) and emodin anthrone, beside two yet unidentified products. No product formation occurred in the absence of acetyl-CoA as starter substrate. To confirm the identities of the enzymatic products, cell-free extracts were incubated with acetyl-CoA and [U-(13)C(3)]malonyl-CoA and (13)C incorporation was analyzed by ESI-MS/MS. Detection of anthranoid biosynthesis in cell-free extracts indicates in vitro cooperation of OKS with a yet unidentified factor or enzyme for octaketide cyclization.
authors: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstrasse 1, 38106 Braunschweig, Germany.
authors: Iman A M
authors: IA
authors: Abdel-Rahman
authors: Till
authors: T
authors: Beuerle
authors: Ludger
authors: L
authors: Ernst
authors: Afaf M
authors: AM
authors: Abdel-Baky
authors: Ezz El-Din K
authors: Eel-D
authors: Desoky
authors: Amany S
authors: AS
authors: Ahmed
authors: Ludger
authors: L
authors: Beerhues
mesh_terms: D000880:Anthraquinones / Q000737:chemistry* / Q000378:metabolism; D002366:Cassia / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018929:Cell Culture Techniques; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D015003:Yeasts
chemical_list: D000880:Anthraquinones; D005656:Fungal Proteins; D048630:Polyketide Synthases