Control of Unsaturation in De Novo Fatty Acid Biosynthesis by FabA.

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.

A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Decoding allosteric regulation by the acyl carrier protein.

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Ploskon et al., Chem. Biol. 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

Synthetic group A streptogramin antibiotics that overcome Vat resistance.

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Molecular basis for interactions between an acyl carrier protein and a ketosynthase.

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and ß-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.

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

At the center of many complex biosynthetic pathways, the acyl carrier protein (ACP) shuttles substrates to appropriate enzymatic partners to produce fatty acids and polyketides. Carrier proteins covalently tether their cargo via a thioester linkage to a phosphopantetheine cofactor. Due to the labile nature of this linkage, chemoenzymatic methods have been developed that involve replacement of the thioester with a more stable amide or ester bond. We explored the importance of the thioester bond to the structure of the carrier protein by using solution NMR spectroscopy and molecular dynamics simulations. Remarkably, the replacement of sulfur with other heteroatoms results in significant structural changes, thus suggesting more rigorous selections of isosteric substitutes is needed.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL.

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems.

Using modern tools to probe the structure-function relationship of fatty acid synthases.

Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in nature, preserving the same handful of chemical reactions across 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, due in part 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 will focus first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. Although significant unknowns remain, new understandings of the intricacies of FAS point to future advances in manipulating this complex molecular factory.

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering.

Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases' many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

Modeling linear and cyclic PKS intermediates through atom replacement.

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.

Trapping the dynamic acyl carrier protein in fatty acid biosynthesis.

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling 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. These techniques, in combination with molecular dynamics simulations, 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 may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. 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 have major roles in the treatment of cancer, obesity and infectious disease.

Sulfonyl 3-alkynyl pantetheinamides as mechanism-based cross-linkers of acyl carrier protein dehydratase.

Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein-protein interactions of ACPs 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 that can cross-link ACPs with catalytic ß-hydroxy-ACP dehydratase (DH) domains by means of 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 noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy 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 cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.