Dataset Information

The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis.

ABSTRACT: The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP(Pks13)). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP(Pks13)) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce alpha-alkyl beta-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.

SUBMITTER: Gavalda S

PROVIDER: S-EPMC2740550 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis.

Gavalda Sabine S Léger Mathieu M van der Rest Benoît B Stella Alexandre A Bardou Fabienne F Montrozier Henri H Chalut Christian C Burlet-Schiltz Odile O Marrakchi Hedia H Daffé Mamadou M Quémard Annaïk A

The Journal of biological chemistry 20090512 29

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests an ...[more]

PMID: 19436070

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Project description:Background: Among the nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 was identified as the condensase required for the final condensation step of two long acyl chains. This leads to mycolic acids, which are essential components of the cell envelope of Corynebacterineae species. The single polypeptide chain of Pks13 comprises five catalytic domains, including two functional acyl carrier protein domains, separated by linkers comprising up to ca. 200 amino acid residues. In many aspects, Pks13 is an atypical polyketide synthase. It has been validated as a promising druggable target and its structure is badly needed to speed up drug discovery to fight against tuberculosis. Results: We report here a quasi-atomic model of Pks13 obtained using small angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. In addition, the structural changes induced by the loading of a substrate analogue have been challenged. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights an identical and elongated structure of the apo and holo forms at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. Apo-Pks13 is in our conditions in a monomeric state, but loading of a substrate analogue push the enzyme towards the functional dimeric state. Conclusions: The Pks13 model provides the first structural information on the molecular mechanism of this complex enzyme and open up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

Project description:UnlabelledMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates.ImportanceMycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Dataset Information

The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis.

Publications

The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis.

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