Abstract
Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4'-phosphopantetheine (Ppant) to create the holo form, (15)N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP.
Highlights
Type I modular polyketide synthases (PKSs) are large, multi-domain complexes responsible for generating natural products with a spectrum of medically important activities, including antibiotic, anticancer, antifungal, antitumour and immunosuppressive properties [1]
Following purification using nickel affinity chromatography, the fusion tag was removed by thrombin cleavage, leaving four non-native amino acid residues originating from the expression vector (GSHM-) at the N-terminus of the construct
Eighty-three of the 84 expected backbone amide resonances were assigned; no signal was detected for His2077, which is located in a flexible surface exposed loop
Summary
Type I modular polyketide synthases (PKSs) are large, multi-domain complexes responsible for generating natural products with a spectrum of medically important activities, including antibiotic, anticancer, antifungal, antitumour and immunosuppressive properties [1]. PKS extension modules comprise three domains essential for constructing the product chain [2]: a small (∼10 kDa) acyl carrier protein (ACP) to which the polyketide substrate is tethered via thioester linkage to a 4 -phosphopantetheine (Ppant) prosthetic group; an acyltransferase (AT), which selects an appropriate extender unit (commonly malonate or methylmalonate as their coenzyme A thioesters) for loading on to the ACP; and a ketosynthase (KS), which accepts a polyketide chain from a previous module and attaches the new extender unit by catalysing a decarboxylative condensation reaction (Figure 1A). To introduce chemical diversity into the polyketide product, modules can contain additional enzyme domains [2]: a ketoreductase (KR) that reduces the β-ketone group to an alcohol and may epimerize the adjacent α-centre; a dehydratase (DH), which eliminates the β-hydroxy to form an α-β double bond; and an enoyl reductase (ER) that reduces the resulting alkene, producing a saturated β-methylene group (Figure 1A). The length and functionality of the final product is defined by the number, order and domain composition of modules within the system [1]
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