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Solution study of a type I polyketide synthase from Mycobacterium tuberculosis: pks13, the condensase of mycolic acids

ABSTRACT: 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.

INSTRUMENT(S): Synapt MS, Orbitrap Fusion

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Julien Marcoux

LAB HEAD: Julien Marcoux

PROVIDER: PXD029754 | Pride | 2022-05-11

REPOSITORIES: Pride

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Project description:Mycobacterium polyketide synthase 13 (Pks13) is a protein complex comprised of a closely symmetric parallel dimer of chains, each encoding several enzymatic and transport functions. This module carries out the condensation of two different very long chain fatty acids to produce mycolic acids that are essential components of the mycobacterial cell wall. Here, we use cryo-EM, XL-MS, and integrative modeling to determine the structure and domain trajectories of the dimeric multi-enzyme Pks13 purified endogenously from mycobacteria under normal growth conditions. Structures of the multi-domain assembly revealed by cryogenic electron microscopy (cryoEM) define the ketosynthase (KS), linker, and acyltransferase (AT) domains, each at atomic resolution (1.8Å), with bound substrates defined at 2.4Å and 2.9Å resolution. Image classification reveals two distinct structures with alternate locations of the N-terminal acyl carrier protein (termed ACP1a, ACP1b) seen at 3.6Å and 4.6Å resolution respectively. These two structures suggest plausible intermediate states, related by a ~60Å movement of ACP1, on the pathway for substrate delivery from the fatty acyl-ACP ligase (FadD32) to the ketosynthase domain. The linking sequence between ACP1 and the KS includes an 11 amino acid sequence with 6 negatively charged side chains that lies in different positively charged grooves on the KS in ACP1a versus ACP1b structures. This charge complementarity between the extended chain and the grooves suggests some stabilization of these two distinct orientations. Other domains are visible at lower resolution and indicate flexibility relative to the KS-AT core. The chemical structures of three bound endogenous long chain fatty acid substrates with their proximal regions defined in the structures were determined by electrospray ionization mass spectrometry. The domain proximities were probed by chemical cross-linking and identified by mass spectrometry (XL-MS). These were incorporated into integrative structure modeling to define multiple domain configurations that transport the very long fatty acid chains throughout the multistep Pks13 mediated synthetic pathway.

			Action	DRS
	F115291.dat	Other
	F115292.dat	Other
	F115293.dat	Other
	F115294.dat	Other
	F115295.dat	Other

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Solution study of a type I polyketide synthase from Mycobacterium tuberculosis: pks13, the condensase of mycolic acids

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