Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13
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ABSTRACT:
SUBMITTER: Robert Michael Stroud
PROVIDER: EMPIAR-11608 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13.
Kim Sun Kyung SK Dickinson Miles Sasha MS Finer-Moore Janet J Guan Ziqiang Z Kaake Robyn M RM Echeverria Ignacia I Chen Jen J Pulido Ernst H EH Sali Andrej A Krogan Nevan J NJ Rosenberg Oren S OS Stroud Robert M RM
Nature structural & molecular biology 20230213 3
The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified fr ...[more]
Similar Datasets
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.
2023-06-13 | PXD033471 | Pride
Project description:Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.
| S-EPMC4278352 | biostudies-literature
Project description:The dehydratases (DHs) of modular polyketide synthases (PKSs) catalyze dehydrations that occur frequently in the biosynthesis of complex polyketides, yet little is known about them structurally or mechanistically. Here, the structure of a DH domain, isolated from the fourth module of the erythromycin PKS, is presented at 1.85 A resolution. As with the DH of the highly related animalian fatty acid synthase, the DH monomer possesses a double-hotdog fold. Two symmetry mates within the crystal lattice make a contact that likely represents the DH dimerization interface within an intact PKS. Conserved hydrophobic residues on the DH surface indicate potential interfaces with two other PKS domains, the ketoreductase and the acyl carrier protein. Mutation of an invariant arginine at the hypothesized acyl carrier protein docking site in the context of the erythromycin PKS resulted in decreased production of the erythromycin precursor 6-deoxyerythronolide B. The structure elucidates how the alpha-hydrogen and beta-hydroxyl group of a polyketide substrate interact with the catalytic histidine and aspartic acid in the DH active site prior to dehydration.
| S-EPMC3496180 | biostudies-literature
Project description:Ketoreductases (KRs) from modular polyketide synthases (PKSs) can perform stereospecific catalysis, selecting a polyketide with a D- or L-?-methyl substituent for NADPH-mediated reduction. In this report, molecular dynamics (MD) simulations were performed to investigate the interactions that control stereospecificity. We studied the A1-type KR from the second module of the amphotericin PKS (A1), which is known to be stereospecific for a D-?-methyl-substituted diketide substrate (dkD). MD simulations of two ternary complexes comprised of the enzyme, NADPH, and either the correct substrate, dkD, or its enantiomer (dkL) were performed. The coordinates for the A1/NADPH binary complex were obtained from a crystal structure (PDB entry 3MJS), and substrates were modeled in the binding pocket in conformations appropriate for reduction. Simulations were intended to reproduce the initial weak binding of the polyketide substrate to the enzyme. Long (tens of nanoseconds) MD simulations show that the correct substrate is retained in a conformation closer to the reactive configuration. Many short (up to a nanosecond) MD runs starting from the initial structures display evidence that Q364, three residues N-terminal to the catalytic tyrosine, forms a hydrogen bond to the incorrect dkL substrate to yield an unreactive conformation that is more favorable than the reactive configuration. This interaction is not as strong for dkD, as the D-?-methyl substituent is positioned between the glutamine and the reactive site. This result correlates with experimental findings [Zheng, J., et al. (2010) Structure 18, 913-922] in which a Q364H mutant was observed to lose stereospecificity.
| S-EPMC4501267 | biostudies-literature
Project description:Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.
| S-EPMC3460465 | biostudies-literature
Project description:Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex known enzymes from secondary metabolism and are responsible for the biosynthesis of highly diverse bioactive polyketides. However, most of these metabolites remain uncharacterized, since trans-AT PKSs frequently occur in poorly studied microbes and feature a remarkable array of non-canonical biosynthetic components with poorly understood functions. As a consequence, genome-guided natural product identification has been challenging. To enable de novo structural predictions for trans-AT PKS-derived polyketides, we developed the trans-AT PKS polyketide predictor (TransATor). TransATor is a versatile bio- and chemoinformatics web application that suggests informative chemical structures for even highly aberrant trans-AT PKS biosynthetic gene clusters, thus permitting hypothesis-based, targeted biotechnological discovery and biosynthetic studies. We demonstrate the applicative scope in several examples, including the characterization of new variants of bioactive natural products as well as structurally new polyketides from unusual bacterial sources.
| S-EPMC6642696 | biostudies-literature
Project description:Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.
| S-EPMC1978548 | biostudies-literature
Project description:The highly reducing iterative polyketide synthases responsible for lovastatin biosynthesis contains a section homologous to condensation (CON) domain observed in nonribosomal peptide synthetases (NRPSs). In the present study, we expressed the isolated lovastatin CON domain and solved the crystal structure to 1.79?Å resolution. The overall structure shows similarity to canonical condensation domains of NRPSs, containing the N-terminal and C-terminal subdomains that resemble enzymes of chloramphenicol acetyltransferase family, whereas distinct structural features are observed at the active site. The acceptor entry of the substrate channel is blocked by a flexible loop, thereby preventing the loading of substrate for a new round of chain elongation. The mutation of conserved catalytic motif located at the midpoint of substrate channel agrees with the incapability of CON to catalyzed amide-bond formation. The structure helps to understand the function of CON in lovastatin biosynthesis.
| S-EPMC6260248 | biostudies-literature
Project description:Polyketide synthases (PKSs) are versatile C-C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains-well characterized on their own-are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide B synthase by small-angle X-ray scattering and cross-linking mass spectrometry with coarse-grained structural modeling. The results of this hybrid approach shed light on the solution structure of a cis-AT type PKS module as well as its inherent conformational dynamics. Supported by a directed evolution approach, we also find that acyl carrier protein (ACP)-mediated substrate shuttling appears to be steered by a nonspecific electrostatic interaction network.
| S-EPMC8717363 | biostudies-literature
Project description:Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the leading causes of death due to infectious disease. The Mammalian Cell Entry proteins are essential virulence factors during infection and are thought to facilitate the import of nutrients across the highly complex mycobacterial cell envelope. We purified an endogenous Mammalian Cell Entry complex from the model mycobacterium, Mycobacterium smegmatis, identified components of the Mce1 protein complex using mass spectrometry, and determined its structure using single-particle cryo-electron microscopy and AlphaFold2. This entry contains the mass spectrometry raw files and search results for the LC-MS analysis of the purified complex.
2022-11-29 | MSV000090807 | MassIVE