The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis.
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.
Gavalda
Sabine
S
CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Départements Mécanismes Moléculaires des Infections Mycobacteriennes, Biologie du Cancer, 205 route de Narbonne, F-31077 Toulouse, France.
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
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D053898:Biosynthetic Pathways; D002853:Chromatography, Liquid; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D009171:Mycolic Acids / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D009171:Mycolic Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D048630:Polyketide Synthases; D003066:Coenzyme A Ligases