Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

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 selectivity in a highly reducing type II polyketide synthase.

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.

Screening and characterization of polyhydroxyalkanoate granules, and phylogenetic analysis of polyhydroxyalkanoate synthase gene PhaC in cyanobacteria.

Using Nile Red and BODIPY 493/503 dye-staining and fluorescence microscopy, twenty cyanobacterial strains, including ten commercially available strains and ten environmental isolates from estuaries, freshwater ponds, and lagoons, were screened for the accumulation of ecologically important and potentially biotechnologically significant carbon storage granules such as polyhydroxyalkanoates (PHA). Dye-staining granules were observed in six strains. Three Synechocystis, spp. strains WHSYN, LSNM, and CGF-1, and a Phormidium-like sp. CGFILA were isolated from environmental sources and found to produce granules of polyhydroxyalkanoate (PHA) according to PHA synthase gene (phaC) PCR screening and 1 H NMR analyses. The environmental isolate, Nodularia sp. Las Olas and commercially available Phormidium cf. iriguum CCALA 759 displayed granules but screened negative for PHA according to phaC PCR and 1 H NMR analyses. Partial polyhydroxyalkanoate synthase subunit C (phaC) and 16S rRNA gene sequences obtained from the PHA-accumulating strains and analyzed alongside publicly available phaC, phaE, 16S rRNA, and 23S rRNA data help in understanding the distribution and evolutionary history of PHA biosynthesis within the phylum Cyanobacteria. The data show that the presence of phaC is highly conserved within the genus Synechocystis, and present in at least one isolate of Phormidium. Maximum likelihood analyses and cophylogenetic modeling of PHA synthase gene sequences provide evidence of a recent horizontal gene transfer event between distant genera of cyanobacteria related to Pleurocapsa sp. PCC 7327 and Phormidium-like sp. CGFILA. These findings will help guide additional screening for PHA producers, and may explain why some Phormidium species produce PHAs, while others do not.

Type II non-ribosomal peptide synthetase proteins: structure, mechanism, and protein-protein interactions.

Covering: 1990 to 2019 Many medicinally-relevant compounds are derived from non-ribosomal peptide synthetase (NRPS) products. Type I NRPSs are organized into large modular complexes, while type II NRPS systems contain standalone or minimal domains that often encompass specialized tailoring enzymes that produce bioactive metabolites. Protein-protein interactions and communication between the type II biosynthetic machinery and various downstream pathways are critical for efficient metabolite production. Importantly, the architecture of type II NRPS proteins makes them ideal targets for combinatorial biosynthesis and metabolic engineering. Future investigations exploring the molecular basis or protein-protein recognition in type II NRPS pathways will guide these engineering efforts. In this review, we consolidate the broad range of NRPS systems containing type II proteins and focus on structural investigations, enzymatic mechanisms, and protein-protein interactions important to unraveling pathways that produce unique metabolites, including dehydrogenated prolines, substituted benzoic acids, substituted amino acids, and cyclopropanes.

Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases.

Metabolic engineering of fatty acids and polyketides remains challenging due to unresolved protein-protein interactions that are essential to synthase activity. While several chemical probes have been developed to capture and visualize protein interfaces in these systems, acyl carrier protein (ACP) transacylase (AT) domains remain elusive. Herein, we combine a mutational strategy with fluorescent probe design to expedite the study of AT domains from fatty acid and polyketide synthases. We describe the design and evaluation of inhibitor-inspired and substrate-mimetic reporters containing sulfonyl fluoride and ß-lactone warheads. Moreover, specific active-site labeling occurs by optimizing pH, time, and probe concentration, and selective labeling is achieved in the presence of inhibitors of competing domains. These findings provide a panel of AT-targeting probes and set the stage for future combinatorial biosynthetic and drug discovery initiatives.

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.

Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate ß-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor salicyl-AMS in mice.

Mycobactin biosynthesis in Mycobacterium tuberculosis facilitates iron acquisition, which is required for growth and virulence. The mycobactin biosynthesis inhibitor salicyl-AMS [5'-O-(N-salicylsulfamoyl)adenosine] inhibits M. tuberculosis growth in vitro under iron-limited conditions. Here, we conducted a single-dose pharmacokinetic study and a monotherapy study of salicyl-AMS with mice. Intraperitoneal injection yielded much better pharmacokinetic parameter values than oral administration did. Monotherapy of salicyl-AMS at 5.6 or 16.7 mg/kg significantly inhibited M. tuberculosis growth in the mouse lung, providing the first in vivo proof of concept for this novel antibacterial strategy.

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.

Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition.

Non-ribosomal peptides play a critical role in the clinic as therapeutic agents. To access more chemically diverse therapeutics, non-ribosomal peptide synthetases (NRPSs) have been targeted for engineering through combinatorial biosynthesis; however, this has been met with limited success in part due to the lack of proper protein-protein interactions between non-cognate proteins. Herein, we report our use of chemical biology to enable X-ray crystallography, molecular dynamics (MD) simulations, and biochemical studies to elucidate binding specificities between peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically, we determined X-ray crystal structures of a type II PCP crosslinked to its cognate A domain, PigG and PigI, and of PigG crosslinked to a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures possess large protein-protein interfaces that predominantly feature hydrophobic interactions, with specific electrostatic interactions that orient the substrate for active site delivery. MD simulations of the PCP-A domain complexes and unbound PCP structures provide a dynamical evaluation of the transient interactions formed at PCP-A domain interfaces, which confirm the previously hypothesized role of a PCP loop as a crucial recognition element. Finally, we demonstrate that the interfacial interactions at the PCP loop 1 region can be modified to control PCP binding specificity through gain-of-function mutations. This work suggests that loop conformational preferences and dynamism account for improved shape complementary in the PCP-A domain interactions. Ultimately, these studies show how crystallographic, biochemical, and computational methods can be used to rationally re-engineer NRPSs for non-cognate interactions.

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases.

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

Protein-protein interaction based substrate control in the E. coli octanoic acid transferase, LipB.

Lipoic acid is an essential cofactor produced in all organisms by diverting octanoic acid derived as an intermediate of type II fatty acid biosynthesis. In bacteria, octanoic acid is transferred from the acyl carrier protein (ACP) to the lipoylated target protein by the octanoyltransferase LipB. LipB has a well-documented substrate selectivity, indicating a mechanism of octanoic acid recognition. The present study reveals the precise protein-protein interactions (PPIs) responsible for this selectivity in Escherichia coli through a combination of solution-state protein NMR titration with high-resolution docking of the experimentally examined substrates. We examine the structural changes of substrate-bound ACP and determine the precise geometry of the LipB interface. Thermodynamic effects from varying substrates were observed by NMR, and steric occlusion of docked models indicates how LipB interprets proper substrate identity via allosteric binding. This study provides a model for elucidating how substrate identity is transferred through the ACP structure to regulate activity in octanoyl transferases.

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces.

Gating mechanism of elongating ß-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, ß-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

Role of MyD88 in IL-1ß and Ethanol Modulation of GABAergic Transmission in the Central Amygdala.

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1ß and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1ß and ethanol's modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1ß's actions in reducing postsynaptic GABAA receptor function. Pharmacological inhibition of MyD88 modulates IL-1ß's action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1ß or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors.

Preparation, Assay, and Application of Chlorinase SalL for the Chemoenzymatic Synthesis of S-Adenosyl-l-Methionine and Analogs.

S-adenosyl-l-methionine (SAM) is universal in biology, serving as the second most common cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the methylation of nucleic acids, proteins, and metabolites. Methylation often imparts regulatory control to DNA and proteins, and leads to an increase in the activity of specialized metabolites such as those developed as pharmaceuticals. There has been increased interest in using SAM analogs in methyltransferase-catalyzed modification of biomolecules. However, SAM and its analogs are expensive and unstable, degrading rapidly under physiological conditions. Thus, the availability of methods to prepare SAM in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C5' ribosyl carbon of SAM leading to the formation of 5'-chloro-5'-deoxyadenosine (ClDA) with concomitant displacement of l-methionine. It has been demonstrated that the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this chapter, we describe methods for the preparation of SalL, and the chemoenzymatic synthesis of SAM and SAM analogs from ClDA and l-methionine congeners using SalL. In addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM coupled to DNA, peptide, and metabolite methylation, and to the incorporation of isotopes into alkylated products.

General platform for systematic quantitative evaluation of small-molecule permeability in bacteria.

The chemical features that impact small-molecule permeability across bacterial membranes are poorly understood, and the resulting lack of tools to predict permeability presents a major obstacle to the discovery and development of novel antibiotics. Antibacterials are known to have vastly different structural and physicochemical properties compared to nonantiinfective drugs, as illustrated herein by principal component analysis (PCA). To understand how these properties influence bacterial permeability, we have developed a systematic approach to evaluate the penetration of diverse compounds into bacteria with distinct cellular envelopes. Intracellular compound accumulation is quantitated using LC-MS/MS, then PCA and Pearson pairwise correlations are used to identify structural and physicochemical parameters that correlate with accumulation. An initial study using 10 sulfonyladenosines in Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis has identified nonobvious correlations between chemical structure and permeability that differ among the various bacteria. Effects of cotreatment with efflux pump inhibitors were also investigated. This sets the stage for use of this platform in larger prospective analyses of diverse chemotypes to identify global relationships between chemical structure and bacterial permeability that would enable the development of predictive tools to accelerate antibiotic drug discovery.