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.
Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory.
The acyl carrier protein (ACP) requires post-translational modification with a 4’-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-acyl carrier protein synthetases (AasSs) from different organisms are able to load even, odd and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.
The acyl carrier protein (ACP) plays a central function in acetate biosynthetic pathways, serving as a tether for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and its protein-protein interactions have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides capable of crosslinking ACPs with catalytic β-hydroxyacyl carrier protein dehydratase (DH) domains based upon a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with non-cognate carrier proteins. Finally, we use solution phase protein NMR to demonstrate that sulfonyl-3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This crosslinking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically-crosslinked enzyme complexes, presenting a solution to ongoing structural challenges.
Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.
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