Initiation Of Fatty Acid Biosynthesis Research Articles

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

Design, Synthesis and Antibacterial Activities of Novel Amide Derivatives Bearing Dioxygenated Rings as Potential β-Ketoacyl-acyl Carrier Protein Synthase III (FabH) Inhibitors.

Fatty acid biosynthesis is essential for bacterial survival. Of these promising targets, β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target. FabH would trigger the initiation of fatty acid biosynthesis and it is highly conserved among Gram-positive and -negative bacteria. A series of novel amide derivatives bearing dioxygenated rings were synthesized and developed as potent inhibitors of FabH. These compounds were determined by 1H-NMR, 13C-NMR, MS and further confirmed by crystallographic diffraction study for compound 19. Furthermore, these compounds were evaluated strong broad-spectrum antibacterial activity. Some compounds with potent antibacterial activities were tested for their Escherichia coli (E. coli) FabH inhibitory activity. Especially, compound 19 showed the most potent antibacterial activity with minimum inhibitory concentration (MIC) values of 1.56-3.13 mg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of 2.4 µM. Docking simulation was performed to position compound 19 into the E. coli FabH active site to determine the probable binding conformation.

A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied in vitro Among the β-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would be supported by the viability of the ΔfabH mutant. In this study, we show that the ΔfabH and ΔyiiD mutations were synthetically lethal and that ΔfabH ΔrelA ΔspoT and ΔfabH ΔdksA synthetic lethality was rescued by the heterologous expression of yiiD In the ΔfabH mutant, the expression of yiiD was positively regulated by (p)ppGpp. The growth defect, reduced cell size, and altered fatty acid profile of the ΔfabH mutant and the growth defect of the ΔfabH ΔfabF fabB15(Ts) mutant in oleate- and palmitate-supplemented medium at 42°C were rescued by the expression of yiiD from a multicopy plasmid. Together, these results indicate that the yiiD-encoded function supported initiation of fatty acid biosynthesis in the absence of FabH. We have renamed yiiD as fabYIMPORTANCE Fatty acid biosynthesis is an essential process conserved across life forms. β-Ketoacyl-ACP synthases are essential for fatty acid biosynthesis. FabH is a β-ketoacyl-ACP synthase that contributes to the initiation of fatty acid biosynthesis in Escherichia coli In this study, we present genetic and biochemical evidence that the yiiD (renamed fabY)-encoded function contributes to the biosynthesis of fatty acid in the absence of FabH activity and that under these conditions, the expression of FabY was regulated by the stringent response factors (p)ppGpp and DksA. Combined inactivation of FabH and FabY resulted in growth arrest, possibly due to the loss of fatty acid biosynthesis. A molecule(s) that inhibits the two activities can be an effective microbicide.

The LexA transcription factor regulates fatty acid biosynthetic genes in the cyanobacterium Synechocystis sp. PCC 6803

Specific transcription factors have been identified in various heterotrophic bacterial species that regulate the sets of genes required for fatty acid metabolism. Here, we report that expression of the fab genes, encoding fatty acid biosynthetic enzymes, is regulated by the global regulator LexA in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. Sll1626, an ortholog of the well-known LexA repressor involved in the SOS response in heterotrophic bacteria, was isolated from crude extracts of Synechocystis by DNA affinity chromatography, reflecting its binding to the upstream region of the acpP-fabF and fabI genes. An electrophoresis mobility shift assay revealed that the recombinant LexA protein can bind to the upstream region of each fab gene tested (fabD, fabH, fabF, fabG, fabZ and fabI). Quantitative RT-PCR analysis of the wild type and a lexA-disrupted mutant strain suggested that LexA acts as a repressor of the fab genes involved in initiation of fatty acid biosynthesis (fabD, fabH and fabF) and the first reductive step in the subsequent elongation cycle (fabG) under normal growth conditions. Under nitrogen-depleted conditions, downregulation of fab gene expression is partly achieved through an increase in LexA-repressing activity. In contrast, under phosphate-depleted conditions, fab gene expression is upregulated, probably due to the loss of repression by LexA. We further demonstrate that elimination of LexA largely increases the production of fatty acids in strains modified to secrete free fatty acids.

Study of Schiff-Base-Derived with Dioxygenated Rings and Nitrogen Heterocycle as Potential β-Ketoacyl-acyl Carrier Protein Synthase III (FabH) Inhibitors.

Fatty acid synthesis (FAS) is an essential metabolism during the whole growth and development process of the bacterial. Several key enzymes which involved in this biosynthetic pathway have been considered as useful targets for the development of new antibacterial agents. Among them, β-ketoacyl-acyl carrier protein synthase III (FabH) is the most magnetic target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved of both Gram-positive and Gram-negative bacteria. Following the previous researches, Schiff-based derivatives with dioxygenated rings and N-heterocycle were synthesized in succession, and their biological activities as potential FabH inhibitors were evaluated in this paper. Among these 15 compounds, compound 2E exhibited the best antibacterial activities with minimum inhibitory concentration (MIC) values 1.56-3.13 mg/mL against the tested bacterial strains and showed the most powerful Escherichia coli (E. coli) FabH inhibitory activities with IC50 of 2.1 µM. Also the conceivable binding conformation of placing compound 2E into the E. coli FabH active site was affirmed docking simulation.

Synthesis and antibacterial activity of cinnamaldehyde acylhydrazone with a 1,4-benzodioxan fragment as a novel class of potent β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitor.

Fatty acid biosynthesis is essential for bacterial survival. β-Ketoacyl-acyl carrier protein (ACP) synthase III (FabH), is a particularly attractive antibacterial target, since it is central to the initiation of fatty acid biosynthesis. Three series of 21 cinnamaldehyde acylhydrazone derivatives, A3-9, B3-9, and C3-9, were synthesized and evaluated for FabH-inhibitory activity. Compound B6 showed the most potent biological activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis (minimum inhibitory concentrations (MICs) values: 1.56-3.13 µg/mL) and was comparable with the positive control. Docking simulation by positioning compound B6 in the FabH structure active site was performed to explore the possible binding model.

Deletion of the β-Acetoacetyl Synthase FabY in Pseudomonas aeruginosa Induces Hypoacylation of Lipopolysaccharide and Increases Antimicrobial Susceptibility

The β-acetoacetyl-acyl carrier protein synthase FabY is a key enzyme in the initiation of fatty acid biosynthesis in Pseudomonas aeruginosa. Deletion of fabY results in an increased susceptibility of P. aeruginosa in vitro to a number of antibiotics, including vancomycin and cephalosporins. Because antibiotic susceptibility can be influenced by changes in membrane lipid composition, we determined the total fatty acid profile of the ΔfabY mutant, which suggested alterations in the lipid A region of the lipopolysaccharide. The majority of lipid A species in the ΔfabY mutant lacked a single secondary lauroyl group, resulting in hypoacylated lipid A. Adding exogenous fatty acids to the growth media restored the wild-type antibiotic susceptibility profile and the wild-type lipid A fatty acid profile. We suggest that incorporation of hypoacylated lipid A species into the outer membrane contributes to the shift in the antibiotic susceptibility profile of the ΔfabY mutant.

Microbial production of short-chain alkanes

Increasing concerns about limited fossil fuels and global environmental problems have focused attention on the need to develop sustainable biofuels from renewable resources. Although microbial production of diesel has been reported, production of another much in demand transport fuel, petrol (gasoline), has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway. First, the β-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo. To increase the formation of short-chain fatty acids suitable for subsequent conversion to SCAs in vivo, the activity of 3-oxoacyl-ACP synthase (FabH), which is inhibited by unsaturated fatty acyl-ACPs, was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene; deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for unsaturated fatty acids biosynthesis. A modified thioesterase was used to convert short-chain fatty acyl-ACPs to the corresponding FFAs, which were then converted to SCAs by the sequential reactions of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l(-1) of SCAs consisting of nonane (327.8 mg l(-1)), dodecane (136.5 mg l(-1)), tridecane (64.8 mg l(-1)), 2-methyl-dodecane (42.8 mg l(-1)) and tetradecane (8.9 mg l(-1)), together with small amounts of other hydrocarbons. Furthermore, this platform strain could produce short-chain FFAs using a fadD-deleted strain, and short-chain fatty esters by introducing the Acinetobacter sp. ADP1 wax ester synthase (atfA) and the E. coli mutant alcohol dehydrogenase (adhE(mut)).

Design, synthesis and antimicrobial activities evaluation of Schiff base derived from secnidazole derivatives as potential FabH inhibitors

FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is critically important to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of novel secnidazole derivatives (1–20) were synthesized and fully characterized by spectroscopic methods and elemental analysis. Among these compounds, 6, 8, 11, 13, 14, 16–20 were reported for the first time. These compounds were tested for antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. The compounds inhibitory assay and docking simulation indicated that compound 20 (E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N′-(3,4,5-trimethylbenzylidene)acetohydrazide with MIC of 3.13–6.25μg/mL against the tested bacterial strains was a potent inhibitor of Escherichia coli FabH.

Novel Schiff‐Base‐Derived FabH Inhibitors with Dioxygenated Rings as Antibiotic Agents

Fatty acid biosynthesis plays a vital role in bacterial survival and several key enzymes involved in this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. Of these promising targets, β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target that could trigger the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Designing small molecules with FabH inhibitory activity displays great significance for developing antibiotic agents, which should be highly selective, nontoxic and broad-spectrum. In this manuscript, a series of novel Schiff base compounds were designed and synthesized, and their biological activities were evaluated as potential inhibitors. Among these 21 new compounds, (E)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)hexadecan-1-amine (10) showed the most potent antibacterial activity with a MIC value of 3.89-7.81 μM(-1) against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with an IC(50) value of 1.6 μM. Docking simulation was performed to position compound 10 into the E. coli FabH active site to determine the probable binding conformation.

Design, synthesis and antibacterial activities of vanillic acylhydrazone derivatives as potential β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors

Fatty acid biosynthesis is essential for bacterial survival. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of acylhydrazone derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong broad-spectrum antibacterial activity. Compounds with potent antibacterial activities were tested for their Escherichia coli FabH inhibitory activity. Especially, compound E9 showed the most potent antibacterial activity with MIC values of 0.39–1.56 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC50 of 2.5 μM. Docking simulation was performed to position compound E9 into the E. coli FabH active site to determine the probable binding conformation.

Streptomyces coelicolor RedP and FabH enzymes, initiating undecylprodiginine and fatty acid biosynthesis, exhibit distinct acyl-CoA and malonyl-acyl carrier protein substrate specificities

RedP is proposed to initiate undecylprodiginine biosynthesis in Streptomyces coelicolor by condensing an acyl-CoA with malonyl-ACP and is homologous to FabH that catalyzes the same reaction for initiation of fatty acid biosynthesis. Herein, we report the substrate specificities of RedP and FabH from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC). RedP activity was observed only with a pairing of acetyl-CoA and malonyl-RedQ, consistent with its proposed role in initiating the formation of acetyl-CoA-derived prodiginines. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. FabH demonstrated greater catalytic efficiency for isobutyryl-CoA in comparison with acetyl-CoA using malonyl-FabC, consistent with the observation that in streptomycetes, a broad mixture of fatty acids is synthesized, with those derived from branched-chain acyl-CoA starter units predominating. Diminished FabH activity was also observed using malonyl-RedQ with the same preference for isobutyryl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can produce branched-chain alkyl prodiginines.

Design, synthesis and biological evaluation of urea derivatives from o-hydroxybenzylamines and phenylisocyanate as potential FabH inhibitors

FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of o-hydroxybenzylamines 1– 16 and its corresponding new urea derivatives 17– 32 were synthesized and fully characterized by spectroscopic methods and elemental analysis. This new urea derivatives class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 1-(5-bromo-2-hydroxybenzyl)-1-(4-fluorophenyl)-3-phenylurea ( 18) and 1-(5-bromo-2-hydroxybenzyl)-1-(4-chlorophenyl)-3-phenylurea ( 20) were potent inhibitors of E. coli FabH.

Design, synthesis, and structure–activity relationships of pyrazole derivatives as potential FabH inhibitors

Fatty acid biosynthesis is essential for bacterial survival. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Fifty-six 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 1-(5-(4-fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (12) and 1-(5-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (13) were potent inhibitors of E. coli FabH.

Design, synthesis and biological evaluation of novel thiazole derivatives as potent FabH inhibitors

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram positive and negative bacteria. Three series of Schiff bases containing thiazole template were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 11 and 18 were potent inhibitors of E. coli FabH.

Structural Basis for Different Specificities of Acyltransferases Associated with the Human Cytosolic and Mitochondrial Fatty Acid Synthases

Animals employ two systems for the de novo biosynthesis of fatty acids: a megasynthase complex in the cytosol (type I) that produces mainly palmitate, and an ensemble of freestanding enzymes in the mitochondria (type II) that produces mainly octanoyl moieties. The acyltransferases responsible for initiation of fatty acid biosynthesis in the two compartments are distinguished by their different substrate specificities: the type I enzyme transfers both the acetyl primer and the malonyl chain extender, whereas the type II enzyme is responsible for translocation of only the malonyl substrate. Crystal structures for the type I and II enzymes, supported by in silico substrate docking studies and mutagenesis experiments that alter their respective specificities, reveal that, although the two enzymes adopt a similar overall fold, subtle differences at their catalytic centers account for their different specificities.

QSAR studies on benzoylaminobenzoic acid derivatives as inhibitors of β-ketoacyl-acyl carrier protein synthase III

Fatty acid biosynthesis is essential for most of the bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, β-ketoacyl-ACP synthase III, is a attractive target since it is central to the initiation of fatty acid biosynthesis. Quantitative structure–activity relationship (QSAR) studies have been carried out on a series of benzoylaminobenzoic acid derivatives as potent inhibitors of FabH and antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Neisseria meningitidis and Escherichia coli, which demonstrate FabH inhibitory activity in cell free and whole cell system. The QSAR studies revealed that inhibitory activity increases with increase in hydrophobicity, molar refractivity, aromaticity, and presence of OH group (on x position of the nucleus). On the other side presence of hetero-atoms like N, O, or S at R 1 position of the nucleus decreases the inhibitory activity. The comparison of QSAR between the FabH inhibitory activity and antibacterial activity against S. aureus, S. pneumoniae, S. pyogenes, E. faecalis, N. meningitidis also demonstrates that the hydrophobocity, aromaticity and presence of OH group (on x position of the nucleus) are conducive for the inhibitory activity.

Structure-Based Design, Synthesis, and Study of Potent Inhibitors of β-Ketoacyl-acyl Carrier Protein Synthase III as Potential Antimicrobial Agents

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, beta-ketoacyl-ACP synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Small molecules that inhibit FabH enzymatic activity have the potential to be candidates within a novel class of selective, nontoxic, broad-spectrum antibacterials. Using crystallographic structural information on these highly conserved active sites and structure based drug design principles, a benzoylaminobenzoic acid series of compounds was developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity against Gram-positive and selected Gram-negative organisms.

Characterization of beta-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis.

The Streptomyces glaucescens fabH gene, encoding beta-ketoacyl-acyl carrier protein (beta-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an Mr of 37,000, while gel filtration analysis determined a native Mr of 72,000 +/- 3,000 (mean +/- standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a Km value of 3.66 microM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 microM for acetyl-CoA, 0.71 microM for butyryl-CoA, and 0.41 microM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.

The biosynthesis of n‐[2H7] fatty acids byArthrobacter globiformis from [U‐2H15] octanoic acid

The principal fatty acids present whenArthrobacter globiformis is grown on a glycine medium free of normal fatty acids were found to be the C15 and C16 anteiso fatty acids; only a small amount of the normal fatty acids (C14 and C16) were present. Cells grown on the same medium but supplemented mented with 0.1 mg/ml [U-(2)H(15)]octanoic acid were found to contain an increased amount of the normal fatty acids and these fatty acids were found to be labeled with 7 deuteriums. I concluded that the octanoic acid is degraded by β-oxidation in these cells to [U-(2)H(7)] butyryl-CoA, which then competes with 2-methylbutyryl-CoA for the initiation of fatty acid biosynthesis.