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Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.

ABSTRACT: Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3',4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3'-hydroxylation of 2,4,6-trihydroxybenzophenone and C-O phenol coupling of the resulting 2,3',4,6-tetrahydroxybenzophenone. Relative to the inserted 3'-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5-trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C-O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs.

SUBMITTER: El-Awaad I

PROVIDER: S-EPMC4858744 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.

El-Awaad Islam I Bocola Marco M Beuerle Till T Liu Benye B Beerhues Ludger L

Nature communications 20160505

Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3',4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3'-hydroxylation of 2,4,6-trihydroxybenzophenone and C-O phenol coupling ...[more]

PMID: 27145837

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Project description:Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. In this study, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and biotransformation. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes (paa) located outside the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl coenzyme A (enoyl-CoA) hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it toward the formation of heptacyclic intermediates. TrlB is a 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces spp. Six seven-membered carbocyclic compounds were identified from the trlC, trlD, trlE, and trlF deletion mutants. Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone, and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. The monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to produce DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp.IMPORTANCE Tropolonoids are promising drug lead compounds because of the versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our present study provides clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids in Streptomyces spp. The study on DHT biosynthesis in Streptomyces exhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of the biosynthetic pathway for natural tropolonoid products and provides alternative synthetic biology tools for engineering novel tropolonoids.

Dataset Information

Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.

Publications

Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.

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