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Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.

Publication ,  Journal Article
Kim, SK; Dickinson, MS; Finer-Moore, J; Guan, Z; Kaake, RM; Echeverria, I; Chen, J; Pulido, EH; Sali, A; Krogan, NJ; Rosenberg, OS; Stroud, RM
Published in: bioRxiv
January 28, 2023

Mycobacterium tuberculosis is currently the leading cause of death by any bacterial infection 1 . The mycolic acid layer of the cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are therefore front line targets for antimycobacterial drug development 2,3 . Polyketide synthase 13 (Pks13) is a module comprised of a closely symmetric parallel dimer of chains, each encoding several enzymatic and transport functions, that 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. Consequently individual enzymatic domains of Pks13 are targets for antimycobacterial drug development 4 . To understand this machinery, we sought to determine the structure and domain trajectories of the dimeric multi-enzyme Pks13, a 2×198,426 Dalton complex, from protein purified endogenously from mycobacteria under normal growth conditions, to capture it with normal substrates bound trapped 'in action'. 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. 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.

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bioRxiv

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Publication Date

January 28, 2023

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United States
 

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Kim, S. K., Dickinson, M. S., Finer-Moore, J., Guan, Z., Kaake, R. M., Echeverria, I., … Stroud, R. M. (2023). Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13. BioRxiv. https://doi.org/10.1101/2023.01.27.525930
Kim, Sun Kyung, Miles Sasha Dickinson, Janet Finer-Moore, Ziqiang Guan, Robyn M. Kaake, Ignacia Echeverria, Jen Chen, et al. “Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.BioRxiv, January 28, 2023. https://doi.org/10.1101/2023.01.27.525930.
Kim SK, Dickinson MS, Finer-Moore J, Guan Z, Kaake RM, Echeverria I, et al. Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13. bioRxiv. 2023 Jan 28;
Kim, Sun Kyung, et al. “Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.BioRxiv, Jan. 2023. Pubmed, doi:10.1101/2023.01.27.525930.
Kim SK, Dickinson MS, Finer-Moore J, Guan Z, Kaake RM, Echeverria I, Chen J, Pulido EH, Sali A, Krogan NJ, Rosenberg OS, Stroud RM. Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13. bioRxiv. 2023 Jan 28;

Published In

bioRxiv

DOI

Publication Date

January 28, 2023

Location

United States