FASII Pathway Research Articles

A Streptococcus pyogenes DegV protein regulates the membrane lipid content and limits the formation of extracellular vesicles.

Membranes contain lipids that are composed of fatty acids (FA) and a polar head. Membrane homeostasis is crucial for optimal bacterial growth and interaction with the environment. Bacteria synthesize their FAs via the FASII pathway. Gram-positive bacteria can incorporate exogenous FAs which need to be phosphorylated to become substrate of the lipid biosynthetic pathway. In many species including staphylococci, streptococci and enterococci, this phosphorylation is carried out by the Fak complex, which is composed of two subunits, FakA and FakB. FakA is the kinase. FakB proteins are members of the DegV family, proteins known to bind FAs. Two or three FakB types have been identified depending on the bacterial species and characterized by their affinity for saturated and/or unsaturated FAs. Some species such as Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possess an uncharacterized additional DegV protein. We identify here this DegV member as a fourth FakB protein, named FakB4. The fakB4 gene is co-regulated with FASII genes suggesting an interaction with endogenous fatty acids. fakB4 deletion has no impact on membrane phospholipid composition nor on the percentage of other major lipids. However, the fakB4 mutant strain produced more lipids and more extracellular membrane vesicles than the wild-type strain. This suggests that FakB4 is involved in endogenous FA binding and controls FA storage or catabolism resulting in a limitation of extracellular FA release via membrane vesicles.

Heterologous expression, purification, and partial characterisation of the apicoplast protein 3-oxoacyl-[acyl-carrier-protein] reductase from Toxoplasma gondii

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.

Inhibition of Plasmodium falciparum Fatty Acid Biosynthesis (FAS-II Pathway) by Natural Flavonoids: A Computer-Aided Drug Designing Approach

Inhibition of Plasmodium falciparum Fatty Acid Biosynthesis (FAS-II Pathway) by Natural Flavonoids: A Computer-Aided Drug Designing Approach

The C-terminal end of mycobacterial HadBC regulates AcpM interaction during the FAS-II pathway: a structural perspective.

Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (β-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC.

The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

Mycobacterial crypto-AcpM as a tool to investigate the consequence of drug binding on its key FAS II partner enzyme HadAB

BackgroundMycobacterial FASII pathway is governed by the Protein-Protein Interaction mediated dynamics existent between Acyl Carrier Protein and its partner enzymes. The dehydratase HadAB, involved in the third step of FASII synthesis has remained a key target of drugs like Thiacetazone (TAC) and its consequence on AcpM binding is yet to be deciphered. Owing to the transient nature of these interactions, analysing their implications as a drug target has been exhausting.MethodsIn this context, we have developed an in vitro method to study the effect of thiocarbamide-containing compounds, TAC and SPA0355 (a thiourea analogue) against mycobacterial HadAB. Additionally, by utilizing crypto-ACP (NBD-tagged Acyl Carrier Protein) as a tool of our choice, we attempted at exploring the effect of TAC and SPA0355 on mycobacterial HadAB.ResultsSPA0355 behaves at par with TAC and undergoes activation in the presence of monooxygenase EthA thus, bringing about a covalent modification in HadA subunit of HadAB. The crypto-ACP method provides insights into the altered substrate housing capability in HadAB associated with the impediment of its AcpM mediated functionality; an outcome attributed to the repercussions associated with the binding of the aforementioned thiourea compounds.ConclusionThis investigation has assisted in unveiling a two-step mechanism undertaken by AcpM for interacting with its corresponding partner protein during acyl chain transfer.General significanceThis study highlights the alterations brought about by drug binding in the interplay between ACP and HadAB. Additionally, this work for the first time establishes the role of SPA0355 as a promising drug candidate against dehydratase HadAB.

Targeting Mycobacterium Tuberculosis Enoyl-Acyl Carrier Protein Reductase Using Computational Tools for Identification of Potential Inhibitor and their Biological Activity.

Enoyl-acyl carrier protein reductase (InhA) of type II fatty acid synthase system is involved in the synthesis of mycolic acids which is a major component of the bacterial cell wall. Since they are the key enzymes playing a very significant role in the FASII pathway of the bacterium. In this study, we have developed a workflow for identification of InhA inhibitors by utilizing in silico virtual screening approaches based on various machine learning algorithms followed by pharmacophore based virtual screening. The hits screened from the models were further subjected to molecular docking. Further, based on the XP docking score best twenty compounds were subjected to molecular dynamics study. Finally, nine compounds were shortlisted on the basis of best stable ligand RMSD, c-alpha RMSD, and RMSF plot for biological evaluation studies. Experimental validation of the shortlisted compounds identified one compound JFD01724 having potent inhibitory activity and was able to inhibit the growth of mycobacterium tuberculosis. Further medicinal chemistry efforts may help to improve the inhibitory potency of the identified compound.

FabI (enoyl acyl carrier protein reductase) - A potential broad spectrum therapeutic target and its inhibitors.

Development of new anti-bacterial agents acting upon underexploited targets and thus evading known mechanisms of resistance is the need of the hour. The highly conserved and distinct bacterial fatty acid biosynthesis pathway (FAS-II), presents a validated and yet relatively underexploited target for drug discovery. FabI and its isoforms (FabL, FabK, FabV and InhA) are essential enoyl-ACP reductases present in several microorganisms. In addition, the components of the FAS-II pathway are distinct from the multi-enzyme FAS-I complex found in mammals. Thus, inhibition of FabI and its isoforms is anticipated to result in broad-spectrum antibacterial activity. Several research groups from industry and academic laboratories have devoted significant efforts to develop effective FabI-targeting antibiotics, which are currently in various stages of clinical development for the treatment of multi-drug resistant bacterial infections. This review summarizes all the natural as well as synthetic inhibitors of gram-positive and gram-negative enoyl ACP reductases (FabI). The knowledge of the reported inhibitors can aid in the development of broad-spectrum antibacterials specifically targeting FabI enzymes from S.aureus, S.epidermidis, B.anthracis, B.cereus, E.coli, P. aeruginosa, P. falciparum and M. tuberculosis.

Improved Butanol Production Using FASII Pathway in E. coli.

n-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., car encoding carboxylic acid reductase from Mycobacterium marinum, sfp encoding phosphopantetheinyl transferase from Bacillus subtilis, and adh2 encoding alcohol dehydrogenase from S. cerevisiae, responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for in vivo conversion of butyrate to butanol under aerobic conditions. An E. coli strain harboring car, sfp, and adh2 in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring tesBT gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant E. coli utilizing FASII pathway.

Fatty Acid Biosynthesis in Chromerids.

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0–C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.

Functional characterization of acyl-CoA binding protein in Neospora caninum

BackgroundLipid metabolism is pivotal for the growth of apicomplexan parasites. Lipid synthesis requires bulk carbon skeleton acyl-CoAs, the transport of which depends on the acyl-CoA binding protein (ACBP). In Neospora caninum, the causative agent of neosporosis, the FASII pathway is required for growth and pathogenicity. However, little is known about the fatty acid transport mechanism in N. caninum.MethodsWe have identified a cytosolic acyl-CoA binding protein, with highly conserved amino acid residues and a typical acyl-CoA binding domain in N. caninum. The recombinant NcACBP protein was expressed to verify the binding activities of NcACBP in vitro, and the heterologous expression of NcACBP in Δacbp yeast in vivo. Lipid extraction from ΔNcACBP or the wild-type of N. caninum was analyzed by GC-MS or TLC. Furthermore, transcriptome analysis was performed to compare the gene expression in different strains.ResultsThe NcACBP recombinant protein was able to specifically bind acyl-CoA esters in vitro. A yeast complementation assay showed that heterologous expression of NcACBP rescued the phenotypic defects in Δacbp yeast, indicating of the binding activity of NcACBP in vivo. The disruption of NcACBP did not perturb the parasite’s growth but enhanced its pathogenicity in mice. The lipidomic analysis showed that disruption of NcACBP caused no obvious changes in the overall abundance and turnover of fatty acids while knockout resulted in the accumulation of triacylglycerol. Transcriptional analysis of ACBP-deficient parasites revealed differentially expressed genes involved in a wide range of biological processes such as lipid metabolism, posttranslational modification, and membrane biogenesis.ConclusionsOur study demonstrated that genetic ablation of NcACBP did not impair the survival and growth phenotype of N. caninum but enhanced its pathogenicity in mice. This deletion did not affect the overall fatty acid composition but modified the abundance of TAG. The loss of NcACBP resulted in global changes in the expression of multiple genes. This study provides a foundation for elucidating the molecular mechanism of lipid metabolism in N. caninum.

Antitubercular Triazines: Optimization and Intrabacterial Metabolism

The triazine antitubercular JSF-2019 was of interestdue to its invitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NO⋅ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent invitro efficacy via release of intrabacterial NO⋅ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NO⋅ release and InhA inhibition.

The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection.

Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein (ACP) reductase II (FabK), which catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed that C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using ClosTron mutagenesis and by growth inhibition upon gene silencing with CRISPR interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazole-derived inhibitor (1). The inability of fatty acids to bypass FASII inhibition is likely due to the function of the transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti- C. difficile drugs that treat CDI but avoid collateral damage to the gut microbiota.

Synergistic roles of acyl-CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism.

Toxoplasma gondii relies on apicoplast-localised FASII pathway and endoplasmic reticulum-associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long-chain acyl-CoA (LCACoAs) esters. Functions of Toxoplasma acyl-CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl-CoA-binding protein (TgACBP1) and a sterol carrier protein-2 (TgSCP2) as cytosolic acyl-CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl-CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC-MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. 13 C labelling assay combined with GC-MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T.gondii.

Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK).

Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.

Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase.

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.

Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

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 Escherichiacoli 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.

Crystal structure of dehydratase component HadAB complex of mycobacterial FAS-II pathway

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like β-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.