Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious disease.
Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-β-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono-and didomain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.polyketide biosynthesis | structural biology | aromatase/cyclase T he biosynthesis of type II aromatic polyketide natural products has been extensively investigated because of the versatile pharmacological properties of these compounds (1-7). The type II polyketide synthase (PKS) is composed of dissociated enzymes that are used iteratively and are responsible for the elongation, cyclization, and modification of the polyketide chain ( Fig. 1) (3,4,8,9). The regiospecific cyclization of an acyl carrier protein (ACP)-linked linear poly-β-ketone intermediate is a key transformation catalyzed by type II PKSs. However, the enzymatic mechanism of cyclization remains poorly understood (10-14). Without such knowledge, the polyketide cyclization pattern cannot be predicted; a full understanding of this process at the molecular level is essential for future biosynthetic engineering efforts.In 2008, we reported the crystal structure of the first aromatase/cyclase (ARO/CYC) (TcmN ARO/CYC), which is a singledomain protein (15). On the basis of the structural analysis and mutagenesis results, we proposed that monodomain ARO/CYCs contain an active site and are capable of catalyzing polyketide cyclization and aromatization. Since then, we have performed structural and biochemical studies of two other monodomain ARO/CYCs: WhiE and ZhuI (Fig. 1) (16, 17). These studies provided strong evidence supporting our hypothesis that ARO/ CYC is the site of polyketide cyclization. However, many type II PKSs contain di-domain ARO/CYCs that have two seemingly identical domains (18-23). Why these enzymes require two domains (as opposed to just one) and how t...
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