Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Structural and Biochemical Studies on Klebsiella Pneumoniae Enoyl-ACP Reductase (FabI) Suggest Flexible Substrate Binding Site.

Klebsiella pneumoniae, a bacterial pathogen infamous for antibiotic resistance, is included in the priority list of pathogens by various public health organizations due to its extraordinary ability to develop multidrug resistance. Bacterial fatty acid biosynthesis pathway-II (FAS-II) has been considered a therapeutic drug target for antibacterial drug discovery. Inhibition of FAS-II enzyme, enoyl-acyl carrier protein reductase, FabI, not only inhibits bacterial infections but also reverses antibiotic resistance. Here, we characterized Klebsiella pneumoniae FabI (KpFabI) using complementary experimental approaches including, biochemical, x-ray crystallography, and molecular dynamics simulation studies. Biophysical studies shows that KpFabI organizes as a tetramer molecular assembly in solution as well as in the crystal structure. Enzyme kinetics studies reveal a distinct catalytic property towards crotonyl CoA and reducing cofactor NADH. Michaelis-Menten constant (Km) values of substrates show that KpFabI has higher preference towards NADH as compared to crotonyl CoA. The crystal structure of tetrameric apo KpFabI folds into a classic Rossman fold in which ß-strands are sandwiched between α-helices. A highly flexible substrate binding region is located toward the interior of the tetrameric assembly. Thermal stability assay on KpFabI with its substrate shows that the flexibility is primarily stabilized by cofactor NADH. Moreover, the molecular dynamics further supports that KpFabI has highly flexible regions at the substrate binding site. Together, these findings provide evidence for highly dynamic substrate binding sites in KpFabI, therefore, this information will be vital for specific inhibitors discovery targeting Klebsiella pneumoniae.

Structure-Based Design and Pharmacophore-Based Virtual Screening of Combinatorial Library of Triclosan Analogues Active against Enoyl-Acyl Carrier Protein Reductase of Plasmodium falciparum with Favourable ADME Profiles.

Cost-effective therapy of neglected and tropical diseases such as malaria requires everlasting drug discovery efforts due to the rapidly emerging drug resistance of the plasmodium parasite. We have carried out computational design of new inhibitors of the enoyl-acyl carrier protein reductase (ENR) of Plasmodium falciparum (PfENR) using computer-aided combinatorial and pharmacophore-based molecular design. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) complexation QSAR model was developed for triclosan-based inhibitors (TCL) and a significant correlation was established between the calculated relative Gibbs free energies of complex formation (∆∆Gcom) between PfENR and TCL and the observed inhibitory potencies of the enzyme (IC50exp) for a training set of 20 known TCL analogues. Validation of the predictive power of the MM-PBSA QSAR model was carried out with the generation of 3D QSAR pharmacophore (PH4). We obtained a reasonable correlation between the relative Gibbs free energy of complex formation ∆∆Gcom and IC50exp values, which explained approximately 95% of the PfENR inhibition data: pIC50exp=-0.0544×∆∆Gcom+6.9336,R2=0.95. A similar agreement was established for the PH4 pharmacophore model of the PfENR inhibition (pIC50exp=0.9754×pIC50pre+0.1596, R2=0.98). Analysis of enzyme-inhibitor binding site interactions suggested suitable building blocks to be used in a virtual combinatorial library of 33,480 TCL analogues. Structural information derived from the complexation model and the PH4 pharmacophore guided us through in silico screening of the virtual combinatorial library of TCL analogues to finally identify potential new TCL inhibitors effective at low nanomolar concentrations. Virtual screening of the library by PfENR-PH4 led to a predicted IC50pre value for the best inhibitor candidate as low as 1.9 nM. Finally, the stability of PfENR-TCLx complexes and the flexibility of the active conformation of the inhibitor for selected top-ranking TCL analogues were checked with the help of molecular dynamics. This computational study resulted in a set of proposed new potent inhibitors with predicted antimalarial effects and favourable pharmacokinetic profiles that act on a novel pharmacological target, PfENR.

Do Go Chasing Waterfalls: Enoyl Reductase (FabI) in Complex with Inhibitors Stabilizes the Tetrameric Structure and Opens Water Channels.

The enzyme enoyl-ACP reductase (FabI) is the limiting step of the membrane's fatty acid biosynthesis in bacteria and a druggable target for novel antibacterial agents. The FabI active form is a homotetramer, which displays the highest affinity to inhibitors. Herein, molecular dynamics studies were carried out using the structure of FabI in complex with known inhibitors to investigate their effects on tetramerization. Our results suggest that multimerization is essential for the integrity of the catalytic site and that inhibitor binding enables the multimerization by stabilizing the substrate binding loop (SBL, L:195-200) coupled with changes in the H4/5 (QR interface). We also observed that AFN-1252 (naphtpyridinone derivative) promotes unique conformational changes affecting monomer-monomer interfaces. These changes are induced by AFN-1252 interaction with key residues in the binding sites (Ala95, Tyr146, and Tyr156). In addition, the analysis of water trajectories indicated that AFN-1252 complexes allow more water molecules to enter the binding site than triclosan and MUT056399 complexes. FabI-AFN-1252 simulations show accumulation of water molecules near the Tyr146/147 pocket, which can become a hotspot to the design of novel FabI inhibitors.

Triclosan is the Predominant Antibacterial Compound in Ontario Sewage Sludge.

Sewage treatment plants (STPs) accumulate both antibiotic and nonantibiotic antimicrobial compounds that can select for antibiotic resistant bacteria. Herein, we aimed to identify the predominant antibacterial compounds impacting E. coli from Ontario sewage sludge consisting of thousands of unknown compounds. Among the 10 extracted sludge samples, 6 extracts exerted significant growth inhibition effects in E. coli. A total of 103 compounds were tentatively detected across the 10 sludge samples by suspect screening, among which the bacterial enoyl-ACP reductase (FabI) inhibitor triclocarban was detected at the highest abundance. A hypomorphic FabI knockdown E. coli strain was highly susceptible to the sludge extracts, confirming FabI inhibitors as the primary antibacterial compounds in the sludge. Protein affinity pulldown identified triclosan as the major ligand binding to a His-tagged FabI protein from the sludge, despite the higher abundance of triclocarban in the same samples. Effect-directed analysis was used to determine the contributions of triclosan to the observed antibacterial potencies. Antibacterial effects were only detected in F17 and F18 across 20 fractions, which was consistent with the elution of triclosan and triclocarban in the same two fractions. Further, potency mass balance analysis confirmed that triclosan explained the majority (58-113%) of inhibition effects from sludge extracts. This study highlighted triclosan as the predominant antibacterial compound in sewage sludge impacting E. coli despite the co-occurrence of numerous other antibiotics and nonantibiotics.

A novel antiplasmodial compound: integration of in silico and in vitro assays.

Malaria is a disease caused by Plasmodium genus. which P. falciparum is responsible for the most severe form of the disease, cerebral malaria. In 2018, 405,000 people died of malaria. Antimalarial drugs have serious adverse effects and limited efficacy due to multidrug-resistant strains. One way to overcome these limitations is the use of computational approaches for prioritizing candidates to phenotypic assays and/or in vitro assays against validated targets. Plasmodium falciparum Enoyl-ACP reductase (PfENR) is noteworthy because it catalyzes the rate-limiting step of the biosynthetic pathway of fatty acid. Thus, the study aimed to identify potential PfENR inhibitors by ligand (2D molecular similarity and pharmacophore models) and structure-based virtual screening (molecular docking). 2D similarity-based virtual screening using Tanimoto Index (> 0.45) selected 29,236 molecules from natural products subset available in ZINC database (n = 181,603). Next, 10 pharmacophore models for PfENR inhibitors were generated and evaluated based on the internal statistical parameters from GALAHAD™ and ROC/AUC curve. These parameters selected a suitable pharmacophore model with one hydrophobic center and two hydrogen bond acceptors. The alignment of the filtered molecules on best pharmacophore model resulted in the selection of 10,977 molecules. These molecules were directed to the docking-based virtual screening by AutoDock Vina 1.1.2 program. These strategies selected one compound to phenotypic assays against parasite. ZINC630259 showed EC50 = 0.12 ± 0.018 µM in antiplasmodial assays and selective index similar to other antimalarial drugs. Finally, MM/PBSA method showed stability of molecule within PfENR binding site (ΔGbinding=-57.337 kJ/mol).Communicated by Ramaswamy H. Sarma.

Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan.

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

LZerD webserver for pairwise and multiple protein-protein docking.

Protein complexes are involved in many important processes in living cells. To understand the mechanisms of these processes, it is necessary to solve the 3D structures of the protein complexes. When protein complex structures have not yet been determined by experiment, protein-protein docking tools can be used to computationally model the structures of these complexes. Here, we present a webserver which provides access to LZerD and Multi-LZerD protein docking tools. The protocol provided by the server have performed consistently among the top in the CAPRI blind evaluation. LZerD docks pairs of structures, while Multi-LZerD can dock three or more structures simultaneously. LZerD uses a soft protein surface representation with 3D Zernike descriptors and explores the binding pose space using geometric hashing. Multi-LZerD performs multi-chain docking by combining pairwise solutions by LZerD. Both methods output full-atom docked models of the input proteins. Users can also input distance constraints between interacting or non-interacting residues as well as residues that locate at the interface or far from the interface. The webserver is equipped with a user-friendly panel that visualizes the distribution and structures of binding poses of top scoring models. The LZerD webserver is available at https://lzerd.kiharalab.org.

Biological evaluation, proposed molecular mechanism through docking and molecular dynamic simulation of derivatives of chitosan.

We synthesized Schiff base and its complexes derivatives of chitosan (CS) in order to develop antibiotic compounds based on functionalized-chitosan against gram-positive and gram-negative bacteria. IR, UV-Vis, AFM, SEM, Melting point, X-ray diffraction (XRD), elemental analysis, and 1H NMR techniques were employed to characterize the chemical structures and properties of these compounds. XRD, UV-Vis, and 1H NMR techniques confirmed the formation of Schiff base and its functionalized-chitosan to metals. Subsequently, our antibacterial studies revealed that antibacterial activities of [Zn(Schiff base)(CS)] against S. aureus bacteria increased compared to those of their compounds. In addition, hemolysis test of CS-Schiff base-Cu(II) demonstrated better hemolytic activity than vitamin C, CS-Schiff base, CS-Schiff base-Zn(II), and CS-Schiff base-Ni(II). In a computational strategy, we carried out the optimization of compounds with molecular mechanics (MM+), Semi-emprical (AM1), Abinitio (STO-3G), AMBER, BIO+(CHARMM), and OPLS. Frontier orbital density distributions (HOMO and LUMO), and the optimized computational UV of the compounds were assessed. The optimized computational UV-Vis was similar to the experimental UV-Vis. We applied the docking methods to predict the DNA binding affinity, Staphylococcus aureus enoyl-acyl carrier protein reductase (ENRs), and Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI). Ultimately, the obtained data herein suggested that Schiff base is more selective toward ENRs and saFabI compared to chitosan, its complexes, and metronidazole.

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.

The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI).

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1-3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix-helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome.

Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

Small-Molecule Inhibition of the C. difficile FAS-II Enzyme, FabK, Results in Selective Activity.

Clostridioides difficile infection (CDI) is a leading cause of significant morbidity, mortality, and healthcare-related costs in the United States. After standard therapy, recurrence rates remain high, and multiple recurrences are not uncommon. Causes include treatments employing broad-spectrum agents that disrupt the normal host microbiota, as well as treatment-resistant spore formation by C. difficile. Thus, novel druggable anti-C. difficile targets that promote narrow-spectrum eradication and inhibition of sporulation are desired. As a critical rate-limiting step within the FAS-II bacterial fatty acid synthesis pathway, which supplies precursory component phospholipids found in bacterial cytoplasmic and spore-mediated membranes, enoyl-acyl carrier protein (ACP) reductase II (FabK) represents such a target. FabK is essential in C. difficile (CdFabK) and is structurally and mechanistically distinct from other isozymes found in gut microbiota species, making CdFabK an attractive narrow-spectrum target. We report here the kinetic evaluation of CdFabK, the biochemical activity of a series of phenylimidazole analogues, and microbiological data suggesting these compounds' selective antibacterial activity against C. difficile versus several other prominent gut organisms. The compounds display promising, selective, low micromolar CdFabK inhibitory activity without significantly affecting the growth of other gut organisms, and the series prototype (1b) is shown to be competitive for the CdFabK cofactor and uncompetitive for the substrate. A series analogue (1g) shows maintained inhibitory activity while also possessing increased solubility. These findings represent the basis for future drug discovery efforts by characterizing the CdFabK enzyme while demonstrating its druggability and potential role as a narrow-spectrum antidifficile target.

Structure-based drug design and in vitro testing reveal new inhibitors of enoyl-acyl carrier protein reductases.

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl-acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro-organisms. In this project, we conducted structure-based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC50 values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC50 value of 7.8 µM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.

In silico studies, synthesis and anticancer activity of novel diphenyl ether-based pyridine derivatives.

A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 ± 0.45 µM) and 3p (IC50 10.57 ± 0.54 µM) were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.

Enhancement in anti-tubercular activity of indole based thiosemicarbazones on complexation with copper(I) and silver(I) halides: Structure elucidation, evaluation and molecular modelling.

A series of monomeric tetrahedral complexes of stoichiometry, [MX(HL)(Ph3P)2] (In case of M = Cu, H1L, X = I, 1; Br, 2; Cl, 3; H3L, X = I, 4; Br, 5; Cl, 6; H4L, X = I, 7; Br, 8; Cl, 9 and in case of M = Ag, H1L, X = Cl, 13; Br, 14; H2L, X = Cl, 15, Br 16; H3L, X = Cl, 17, Br, 18) were synthesized by the reaction of copper (I) or silver (I) halides with indole-3-thiosemicarbazone (H1L) or 5-methoxy indole-3-thiosemicarbazone (H2L) or 5-methoxy indole-N1-methyl-3-thiosemicarbazone (H3L), whereas dimers of stoichiometry, [Cu2(µ-X)2(η1-S-H2L)2(Ph3P)2] (X = I, 10; Br, 11; Cl, 12) were obtained by the reaction of copper (I) halides with indole-N1-methyl-3-thiosemicarbazone (HIntsc-N1-Me, H2L). The synthesized complexes were characterized using NMR (1H and 13C) and single crystal X-ray diffraction (H2L, 3, 7, 8, 10, 11 and 13) as well as elemental analysis. Anti- M. tuberculosis activity of ligands (H1L-H4L) and their metal complexes (1-18) were evaluated against M. tuberculosis H37RV strain ATCC 27294. It has been observed that there is unusual enhancement in anti TB activity of these ligands on complexation with copper (I) and silver (I). Molecular modelling studies in the active binding site are also giving complementary theoretical support for the experimental biological data acquired.

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 Šresolution 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.

Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis.

Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric forms of baFabI in the apo state and in complex with NAD+ and with NAD+-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix η7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of baFabI is required for cofactor binding and catalytic activity.

Elucidating Substrate Promiscuity within the FabI Enzyme Family.

The rapidly growing appreciation of enzymes' catalytic and substrate promiscuity may lead to their expanded use in the fields of chemical synthesis and industrial biotechnology. Here, we explore the substrate promiscuity of enoyl-acyl carrier protein reductases (commonly known as FabI) and how that promiscuity is a function of inherent reactivity and the geometric demands of the enzyme's active site. We demonstrate that these enzymes catalyze the reduction of a wide range of substrates, particularly α,ß-unsaturated aldehydes. In addition, we demonstrate that a combination of quantum mechanical hydride affinity calculations and molecular docking can be used to rapidly categorize compounds that FabI can use as substrates. The results here provide new insight into the determinants of catalysis for FabI and set the stage for the development of a new assay for drug discovery, organic synthesis, and novel biocatalysts.

Structural and biochemical characterization of FabK from Thermotoga maritima.

TM0800 from Thermotoga maritima is one of the hypothetical proteins with unknown function. The crystal structure determined at 2.3 Å resolution reveals a two domain structure: the N-terminal domain forming a barrel and the C-terminal forming a lid. One FMN is bound between the two domains with the phosphate making intricate hydrogen bonds with protein and three tightly bound water molecules, and the isoalloxazine ring packed against the side chains of Met22 and Met276. The structure is almost identical to that of FabK (enoyl-acyl carrier protein (ACP) reductase, ENR II), a key enzyme in bacterial type II fatty-acid biosynthesis that catalyzes the final step in each elongation cycle; and the enzymatic activity confirms that TM0800 is an ENR. Enzymatic activity was almost completely abolished when the helices connecting the barrel and the lid were deleted. Also, the Met276Ala and Ser280Ala mutants showed a significant reduction in enzymatic activity. The crystal structure of Met276Ala mutant at 1.9 Å resolution showed an absence of FMN suggesting that FMN plays a role in catalysis, and Met276 is important in positioning FMN. TmFabK exists as a dimer in both solution and crystal. Together this study provides molecular basis for the catalytic activity of FabK.