Dataset Information

Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis.

ABSTRACT: The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.

SUBMITTER: Dorrestein PC

PROVIDER: S-EPMC1236592 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis.

Dorrestein Pieter C PC Yeh Ellen E Garneau-Tsodikova Sylvie S Kelleher Neil L NL Walsh Christopher T CT

Proceedings of the National Academy of Sciences of the United States of America 20050914 39

The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA ...[more]

PMID: 16162666

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Project description:Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

Dataset Information

Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis.

Publications

Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis.

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