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Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis.

ABSTRACT: Melanins are a broad class of darkly pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are known virulence factors that contribute to pathogenicity. Their biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8-tetrahydroxynaphthalene (THN) precursor assembled by multidomain, nonreducing polyketide synthases. Convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that utilize conventional C-terminal thioesterase/Claisen cyclase domains and separate side-chain deacylases. Here, in vitro characterization of Pks1 from Colletotrichum lagenarium establishes a true THN synthase with a bifunctional thioesterase (TE) catalyzing both cyclization and deacetylation of an enzyme-bound hexaketide substrate. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin TEs to gain insight into this unprecedented catalysis of carbon-carbon bond making and breaking by an ?/?-hydrolase fold enzyme.

SUBMITTER: Vagstad AL

PROVIDER: S-EPMC3530136 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis.

Vagstad Anna L AL Hill Eric A EA Labonte Jason W JW Townsend Craig A CA

Chemistry & biology 20121201 12

Melanins are a broad class of darkly pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are known virulence factors that contribute to pathogenicity. Their biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8-tetrahydroxynaphthalene (THN) precursor assembled by multidomain, nonreducing polyketide synthases. Convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that u ...[more]

PMID: 23261597

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Project description:The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel ?-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the ?-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-?-lactamase-type thioesterase (M?L-TE). The bifunctional M?L-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon ?-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of ?-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt M?L-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The M?L-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an ?/? hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

Dataset Information

Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis.

Publications

Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis.

Similar Datasets

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