The structural organization of substrate loading in iterative polyketide synthases

Abstract

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/­condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

The many faces and important roles of protein–protein interactions during non-ribosomal peptide synthesis

“…[126][127][128] A recent and highly important example of the use of this technique to investigate polyketide synthesis was carried out by the Maier and Townsend groups, who could identify and characterise a functionally relevant asymmetric conformation of the protein that was not apparent from crystallographic studies of the same protein. 129 Considering the monomeric nature of NRPS machineries and high degree of variation in module architecture even within one assembly line there is little doubt that cryo-electron microscopy (particularly when coupled with the use of chemical probes to trap the machinery in specic, dened catalytic states) will deliver important contributions to our understanding of NRPS machineries over the years to come. Given the diversity of NRPS systems and their resultant products, it is also conceivable that different NRPS systems will adopt different higher order structures due to the constraints placed on the enzymatic catalysis required to be performed by each individual system.…”

Gating mechanism of elongating β-ketoacyl-ACP synthases

“…atty acid synthases (FASs) and polyketide synthases (PKSs) iteratively condense and modify ketide units to produce a variety of natural compounds, ranging from fatty acids to complex bioactive molecules 1,2 . FASs and PKSs can exist as one or more polypeptide mega-synthases that contain distinct catalytic domains (type I) or as discrete enzymes, each possessing specific activities (type II) [3][4][5][6][7][8] . In these pathways, β-ketoacyl ACP synthases, alternatively ketosynthases (KSs), catalyze carbon-carbon formation via a precisely choreographed decarboxylative Claisen-like condensation to produce a β-ketoacyl species.…”

“…ACPs are initially translated to an inactive apo form and are subsequently post-translationally modified to an active holo form via attachment of a 4′-phosphopantetheine (PPant) arm at a conserved serine residue, providing a thiol moiety that ligates substrates and pathway intermediates to the ACP 10 . Protein-protein interactions (PPIs) between ACPs and their enzymatic partners (or domains) regulate the catalytic activities of FASs and PKSs and additionally provide a mechanism to ensure pathway orthogonality between primary (i.e., FAB) and secondary metabolism (i.e., PKB) 3,5,6,[12][13][14][15][16][17] .…”

Modifying the Thioester Linkage Affects the Structure of the Acyl Carrier Protein

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At the center of many complex biosynthetic pathways,t he acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides.C arrier proteins covalently tether their cargo via at hioester linkage to ap hosphopantetheine cofactor.D ue to the labile nature of this linkage,chemoenzymatic methods have been developed that involve replacement of the thioester with amore stable amide or ester bond. [6][7][8] Using this strategy,crystal structures of E. coli fatty acid ACP( AcpP) crosslinked to FabA, [9] FabB, [10] and FabZ [11] have been determined, which reveal discrete molec-ular interactions that mediate AcpP binding to these enzymes. [1][2][3] Ac lear structural understanding of ACPs and their interactions with partner enzymes is essential to furthering metabolic engineering and drug discovery.E fforts in this regard have proven challenging due to transient interactions and the dynamic nature of the ACP, which transports intermediates to multiple enzymes via ac ovalent but labile thioester linkage.T he archetypical carrier protein AcpP,w hich is involved in type II fatty acid biosynthesis in Escherichia coli,h as been well studied, [4,5] but key questions regarding conformational dynamics and interactions between AcpP and partner enzymes remain partially understood.

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“…Chemoenzymatic preparations of ACPs bearing crosslinking probes have been used to covalently trap ACP-enzyme complexes. [6][7][8] Using this strategy,crystal structures of E. coli fatty acid ACP( AcpP) crosslinked to FabA, [9] FabB, [10] and FabZ [11] have been determined, which reveal discrete molec-ular interactions that mediate AcpP binding to these enzymes. What remains unknown, however, are the dynamics of these interactions throughout ACP-partner protein binding events, including how intermediates attached to the ACPi nfluence protein dynamics and molecular recognition.…”