Acyl Carrier Protein Synthase Research Articles

Computer-aided drug design to generate a unique antibiotic family.

The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 μg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.

Identification of potent indolizine derivatives against Mycobacterial tuberculosis: In vitro anti-TB properties, in silico target validation, molecular docking and dynamics studies

In the current study, two sets of compounds: (E)-1-(2-(4-substitutedphenyl)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium derivatives (3a-3e); and (E)-3-(substitutedbenzoyl)-7-((hydroxyimino)methyl)-2-substitutedindolizine-1-carboxylate derivatives (5a-5j), were synthesized and biologically evaluated against two strains of Mycobacterial tuberculosis (ATCC 25177) and multi-drug resistant (MDR) strains. Further, they were also tested in vitro against the mycobacterial InhA enzyme. The in vitro results showed excellent inhibitory activities against both MTB strains and compounds 5a-5j were found to be more potent, and their MIC values ranged from 5 to 16 μg/mL and 16–64 μg/mL against the M. tuberculosis (ATCC 25177) and MDR-TB strains, respectively. Compound 5h with phenyl and 4-fluorobenzoyl groups attached to the 2- and 3-position of the indolizine core was found to be the most active against both strains with MIC values of 5 μg/mL and 16 μg/mL, respectively. On the other hand, the two sets of compounds showed weak to moderate inhibition of InhA enzyme activity that ranged from 5 to 17 % and 10–52 %, respectively, with compound 5f containing 4-fluoro benzoyl group attached to the 3-position of the indolizine core being the most active (52 % inhibition of InhA). Unfortunately, there was no clear correlation between the InhA inhibitory activity and MIC values of the tested compounds, indicating the probability that they might have different modes of action other than InhA inhibition.Therefore, a computational investigation was conducted by employing molecular docking to identify their putative drug target(s) and, consequently, understand their mechanism of action. A panel of 20 essential mycobacterial enzymes was investigated, of which β-ketoacyl acyl carrier protein synthase I (KasA) and pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (BioA) enzymes were revealed as putative targets for compounds 3a-3e and 5a-5j, respectively. Moreover, in silico ADMET predictions showed adequate properties for these compounds, making them promising leads worthy of further optimization.

Non-modular fatty acid synthases yield distinct N-terminal acylation in ribosomal peptides.

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machinery from other natural product families. Here we report lipoavitides, a class of RiPP/fatty-acid hybrid lipopeptides that display a unique, putatively membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N terminus. The HMP is formed via condensation of isobutyryl-coenzyme A (isobutyryl-CoA) and methylmalonyl-CoA catalysed by a 3-ketoacyl-(acyl carrier protein) synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty-acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

Multidirectional pathway engineering of Mycobacterium neoaurum LY-2 for the enhancement of androstenedione production from phytosterols

Androstenedione (ADD), an important precursor for steroid hormone drug synthesis, can be obtained through the transformation of phytosterols by members of the Mycobacteria genus. In this study, multidirectional pathway engineering of Mycobacterium neoaurum LY-2 was applied to improve ADD production from phytosterols. Initially, the nonessential gene KasB encoding a cell wall β-ketoacyl acyl carrier protein synthase in Mycobacterium neoaurum LY-2 was knocked out, and the production of ADD was increased by 21.0%. Then, the key enzymes involved in ADD synthetic metabolism were systematically explored. These assays revealed that propionyl coenzyme A carboxylase (PCC) and Hsd4A played important roles in the synthesis of ADD. In addition, the heterologous expression of Vitreoscilla hemoglobin (VHb), a protein with oxygen-binding capacity, was successfully realized in Mycobacterium neoaurum LY-2, and the ADD concentration reached 4.42 g/L. Finally, the recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb with high ADD production was constructed by combining the above strategies. Through optimization of conversion conditions, the recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb produced an ADD concentration of 11.12 g/L when adding 20 g/L phytosterols.

N-Heterocyclic Carbene-Catalyzed Facile Synthesis of Phthalidyl Sulfonohydrazones: Density Functional Theory Mechanistic Insights and Docking Interactions.

N-heterocyclic carbene catalysis reaction protocol is disclosed for the synthesis of phthalidyl sulfonohydrazones. A broad range of N-tosyl hydrazones react effectively with phthalaldehyde derivatives under open-air conditions, enabling the formation of a new C-N bond via an oxidative path. The reaction proceeds under mild reaction conditions with broad substrate scopes, wide functional group tolerance, and good to excellent yields. The mechanistic pathway is studied successfully using control experiments, competitive reactions, ESI-MS spectral analyses of the reaction mixture, and computational study by density functional theory. The potential use of one of the phthalidyl sulfonohydrazone derivatives as the inhibitor of β-ketoacyl acyl carrier protein synthase I of Escherichia coli is investigated using molecular docking.

Investigating the initial steps of auricin biosynthesis using synthetic biology

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with d-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and β (KSα, KSβ). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.

Antibacterial, Anticandidal, and Antibiofilm Potential of Fenchone: In Vitro, Molecular Docking and In Silico/ADMET Study.

The aim of the present study is to investigate the effective antimicrobial and antibiofilm properties of fenchone, a biologically active bicyclic monoterpene, against infections caused by bacteria and Candida spp. The interactions between fenchone and three distinct proteins from Escherichia coli (β-ketoacyl acyl carrier protein synthase), Candida albicans (1, 3-β–D-glucan synthase), and Pseudomonas aeruginosa (Anthranilate-CoA ligase) were predicted using molecular docking and in silico/ADMET methods. Further, to validate the in-silico prediction, the antibacterial and antifungal potential of fenchone was evaluated against E. coli, P. aeruginosa, and C. albicans by determining minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and minimum fungicidal concentration (MFC). The lowest MIC/MBC values of fenchone against E. coli and P. aeruginosa obtained was 8.3 ± 3.6/25 ± 0.0 and 266.6 ± 115.4/533.3 ± 230.9 mg/mL, respectively, whereas the MIC/MFC value for C. albicans was found to be 41.6 ± 14.4/83.3 ± 28.8 mg/mL. It was observed that fenchone has a significant effect on antimicrobial activity (p < 0.05). Our findings demonstrated that fenchone at 1 mg/mL significantly reduced the production of biofilm (p < 0.001) in E. coli, P. aeruginosa, and C. albicans by 70.03, 64.72, and 61.71%, respectively, in a dose-dependent manner when compared to control. Based on these results, it has been suggested that the essential oil from plants can be a great source of pharmaceutical ingredients for developing new antimicrobial drugs.

Loss of β-Ketoacyl Acyl Carrier Protein Synthase III Activity Restores Multidrug-Resistant Escherichia coli Sensitivity to Previously Ineffective Antibiotics.

ABSTRACTAntibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, 9 out of 12 are Gram-negative, with four being of “critical priority.” One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes, making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial type II fatty acid synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic-susceptible and antibiotic-resistant clinical isolates. The fabH gene encodes the β-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multidrug-resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens.IMPORTANCE Gram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor toward antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids, and their synthesis constitutes an attractive antimicrobial target, as it follows distinct pathways in prokaryotes and eukaryotes. Here, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are resensitized to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.

Isoniazid: An Exploratory Review

Isoniazid (INH) is one of the most successful tuberculosis medications in the market today. In particular, isoniazid is used as a prophylaxis medication to avoid resurgence of illness in those who have underlying Mycobacterium tuberculosis (MTB) infection. The mode of action of isoniazid is complicated and incorporates a number of distinct aspects in which various biomolecular routes are impacted, including mycolic acid production. Catalase-peroxidase (KatG) activates the prodrug isoniazid and enzymes such as β-ketoacyl ACP synthase (KasA) and enoyl acyl carrier protein (ACP) reductase target the active isoniazid products. Various genes in diverse biochemical networks and pathways are involved in the physiological mechanisms of isoniazid resistance. Isoniazid resistance is the most common of all clinical drug-resistant isolates, with incidence in some areas of up to 20 to 30%. In this review article, several existing components that may influence to the complexities of isoniazid function including mechanism of action, resistance mechanisms in MTB, along with their history, different synthetic procedures, uses, dosage forms, side effects, adverse drug reactions, physico-chemical characteristics, ADME properties, contraindications as well as future perspectives are discussed. Studies of pharmacokinetics have found that the cause of the drug mediated hepatotoxicity is possible by metabolism of isoniazid. Because of inter-individual heterogeneity of polymorphism that affect isoniazid metabolism rates, customized medicines may be required in various populations to prevent hepatotoxicity. The isoniazid multidrug combination treatment which would proved to be effective tuberculosis treatment in future. Further exploration is needed for better comprehension of pathogenesis mechanism of Mycobacterium tuberculosis (MTB) and drug resistance studies are required for building up better therapeutics and diagnostic against tuberculosis.

Initiation of Fatty Acid Synthesis by a Malonyl‐ACP Decarboxylase

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. In Escherichia coli FabH (3-ketoacyl acyl carrier protein (ACP) synthase III) initiates fatty acid synthesis by condensing acetyl coenzyme A (acetyl-CoA) with malonyl-ACP. This work describes a new pathway enzyme, malonyl-ACP decarboxylase (MadA), that permits ΔfabH knockout strains to survive by providing an alternate pathway for the initiation of fatty acid synthesis. MadA has an N-terminal GCN5-related N-acetyltransferase (GNAT) domain and a C-terminal hotdog domain. The full-length protein, the N-terminal (MadAN) and C-terminal (MadAC) domains were purified. MadA and MadAC are dimers and MadAN is a monomer. MadAC is a malonyl-ACP decarboxylase and MadAN is not. Mad proteins are widely distributed in Proteobacteria and most contain only the C-terminal hot dog dimerization and catalytic domain (MadB). The MadB homolog in Shewanella oneidensis was purified. Site directed mutagenesis guided by the MadB crystal structure was used to identify the active site loop. Asn45 has a critical role in the malonyl-ACP decarboxylase activity. MadA initiates FabH-independent fatty acid synthesis by decarboxylating malonyl-ACP to acetyl-ACP. FabB/F then condenses malonyl-ACP and acetyl-ACP to acetoacetyl-ACP, which enters the elongation cycle. MadAC and MadB both restore the cell size defect in the ΔfabH strain.

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy.

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca2+ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α1α2 loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α2 and α3-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α2-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

BackgroundPlipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.ResultsThe 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.ConclusionsThis study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Identification of inhibitor binding hotspots in Acinetobacter baumannii β-ketoacyl acyl carrier protein synthase III using molecular dynamics simulation

Acinetobacter baumannii is a gram-negative bacterium that is rapidly developing drug resistance due to the abuse of antibiotics. The emergence of multidrug-resistantA. baumannii has greatly contributed to the urgency of developing new antibiotics. Previously, we had discovered two potent inhibitors of A. baumannii β-ketoacyl acyl carrier protein synthase III (abKAS III), YKab-4 and YKab-6, which showed potent activity against A. baumannii. In addition, we have reported the crystal structure of abKAS III. In the present study, we investigated the binding between abKAS III and its inhibitors by docking simulation. Molecular dynamics (MD) simulations were performed using docked inhibitor models to identify the hotspot residues related to inhibitor binding. The binding free energies estimated using the MD simulations suggest that residues I198 and F260 of abKAS III serve as the inhibitor binding hotspots. I198, found to be responsible for mediating hydrophobic interactions with inhibitors, had the strongest residual binding energy among all abKAS III residues. We modeled glutamine substitutions of residues I198 and F260 and estimated the relative binding energies of the I198Q and F260Q variants. The results confirmed that I198 and F260 are the key inhibitor binding residues. The roles of the key residues in inhibitor binding, i.e. F260 in the α9 helix and the I198 in the β6β7 loop region, were investigated using principal component analysis (PCA). PCA revealed the structural changes resulting from the abKAS III I198Q and F260Q mutations and described the essential dynamics of the α9 helix. In addition, the results suggest that the β6β7 loop region may act as a gate keeper for ligand binding. Hydrophobic interactions involving I198 and F260 in abKAS III appear to be essential for the binding of the inhibitors YKab-4 and YKab-6. In conclusion, this study provides valuable information for the rational design of antibiotics via the inhibition of abKAS III.

Constructing Combinatorial Synthases Using Acyl Carrier Protein Chimeras

Synthases encoded by biosynthetic gene clusters in microorganisms are capable of producing structurally complex molecules of diverse function. The ability to mix‐and‐match enzymes from different synthases to create “unnatural natural products” could enable the environmentally‐responsible construction of new pharmaceutically relevant molecules. Acyl carrier proteins (ACPs) are central hubs within biosynthetic pathways as they mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural product. Past combinatorial biosynthesis efforts have failed because of incompatible ACP‐enzyme interactions. To overcome this obstacle, chimeric ACPs capable of cross‐talk between different biosynthetic pathways were designed to serve as a lynchpin in functional hybrid synthases. Chimeric ACPs were constructed by combining features of the actinorhodin polyketide synthase ACP (ACT ACP) and of E. coli fatty acid synthase ACP (AcpP). Given the premise that AcpP interacts with its cognate ketosynthase, FabF, whereas ACT ACP does not, the ability of chimeric ACPs to form a mechanistically‐relevant crosslink with FabF was evaluated. A colorimetric assay was used to assess ACP interactions and revealed the role of Helix II of AcpP in driving ACP‐FabF binding. Results suggest that the residues guiding ACP‐FabF compatibility may reside in Helix II. Future directions will include using site directed mutagenesis to investigate the specific residues responsible for ACP‐FabF recognition.Support or Funding InformationNational Institutes of Health (R15GM120704 to L.K.C)National Science Foundation CAREER Award (CHE‐1652424 to L.K.C.)Cottrell Scholars Award (24350 to L.K.C.)

Alternate fatty acid synthesis initiation in Escherichia coli

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. FabH (3‐ketoacyl acyl carrier protein (ACP) synthase III) initiates fatty acid synthesis by condensing acetyl coenzyme A (acetyl‐CoA) with malonyl‐ACP in Escherichia coli. FabH was long thought to be the sole enzyme responsible for initiation of FASII and therefore essential for survival. The observation that fabH deletion strains are viable suggested the existence of an alternate initiation pathway. The yiiD gene was recently identified as essential for initiation of fatty acid synthesis in the absence of FabH in E. coli. The biochemical activity of YiiD is not known.YiiD has an N‐terminal GCN5‐related N‐acetyltransferase (GNAT) domain and a C‐terminal hotdog domain that is annotated as a thioesterase. The full‐length protein, as well as the N‐terminal and C‐terminal domains were purified using an E. coli expression system. Full length YiiD and the C‐terminal hotdog domain are dimers and the N‐terminal GNAT domain is a monomer.YiiD lacks condensing enzyme activity, suggesting that the initiation of FASII by YiiD is occurring through an alternate mechanism. We hypothesized that YiiD is initiating FASII by a transacylase reaction. FabH has acetyl‐CoA:ACP transacetylase activity, however protein extracts from an E. coli fabH deletion strain retain transacetylase activity. Purified YiiD has acetyl‐CoA:ACP transacetylase activity. The N‐terminal GNAT domain was inactive, whereas the C‐terminal domain was 100‐fold more active than intact YiiD. This suggests that the C‐terminal domain has the transacetylase active site and that the N‐terminal domain negatively regulates this activity. These data are consistent with YiiD forming acetyl‐ACP that is used by the elongation condensing enzymes (FabB or FabF) to initiate FASII thus bypassing FabH.Support or Funding InformationNational Institutes of Health Grant GM034496, Cancer Center (CORE) Support Grant CA21765, American and Lebanese Syrian Associated Charities (ALSAC)Proposed model for alternate initiation of fatty acid synthesis by YiiD in E. coliFigure 1

High temperature during soybean seed development differentially alters lipid and protein metabolism

ABSTRACT High temperatures during seed development can affect the seed yield and quality in many crops. Here, we analyzed how high temperature alters the main seed storage compounds (lipid and protein) in soybean. At five days after R5 stage (initial seed filling stage), soybean plants were treated with control (20/20ºC day/night) and high temperature (30/30ºC day/night). After treatment, immature seed was sampled, analyzed for lipid and protein contents and for expression of seed storage compounds related genes. High temperature during seed filling increased lipid content but decreased protein content, associating with yield reduction. It increased the expression of two genes related to seed lipid biosynthesis (GmBCCP2 and GmKAS1) and genes for a lipid biosynthesis regulator (GmWRI1) and its transcription factor (GmDREBL), and decreased the expression of genes related to lipid degradation such as GmACXs. High temperature downregulated genes related to seed storage protein (GmGy1, GmGy2, GmGy4, GmGy5 and Gmβ-conglycinin) and upregulated genes for cysteine and aspartate proteinases. Therefore, high temperature during seed filling preferentially accumulates lipid than protein content in seed, although seed yield reduction was associated with lower seed protein content in soybean. Our study provides insights for further improvements of soybean seed oil under abiotic stress such as heat stress. Abbreviations: DAT: days after treatment; TF: transcription factor; DREBL: Dehydration-responsive Element-binding L; WRI1: Wrinkled 1; PK: Ketoacyl acyl carrier protein synthase 1; PEP: phosphoenolpyruvate; BCCP2: Biotin carboxyl carrier protein; KAS1: ketoacyl acyl carrier protein synthase 1; ACX: Acyl-coenzyme A peroxidase; MS: Malate synthase; PEPCK: Phosphoenolpyruvate carboxykinase; Gy: Glycinin; CysP: Cysteine Proteinase; SoyAP: Aspartic Proteinase; EF1b: Elongation factor 1b.

Chloroplastic acyl carrier protein synthase I and chloroplastic 20 kDa chaperonin proteins are involved in wheat (Triticum aestivum) in response to moisture stress

ABSTRACT In this study, two bread wheat (Triticum aestivum L.) cultivars, Pishgam (drought-tolerant) and Shahryar (drought-sensitive), were grown in the greenhouse under control and moisture stress conditions. Based on phenological and morpho-physiological results, Pishgam was confirmed as a moisture stress tolerant cultivar. In the fallowing, at the start of heading time, its treated and untreated flag leaves were sampled for two-dimensional electrophoresis (2-DE) based on proteomics approach. Among approximately 263 protein spots appearing in two-dimensional gels, 23 and 10 protein spots were up- and down-regulated, respectively. Among these differentially expressed proteins, 11 proteins with more differences were identified by MALDI TOF/TOF MS which allocated to six functional protein groups involved in photosynthesis or respiration, carbohydrate metabolism, energy metabolism, chaperon, lipid metabolism and unknown function. We report this for the first time that chloroplastic acyl carrier protein synthase I and chloroplastic 20 kDa chaperonin proteins were significantly changed in wheat in response to moisture stress.

Synthesis of Disulfide-Bridged N-Phenyl-N'-(alkyl/aryl/heteroaryl)urea Derivatives and Evaluation of Their Antimicrobial Activities.

The discovery of new antimicrobial agents is extremely needed to overcome multidrug-resistant bacterial and tuberculosis infections. In the present study, eight novel substituted urea derivatives (10a-10h) containing disulfide bond were designed, synthesized and screened for their in vitro antimicrobial activities on standard strains of Gram-positive and Gram-negative bacteria as well as on Mycobacterium tuberculosis. According to the obtained results, antibacterial effects of the compounds were found to be considerably better than their antimycobacterial activities along with their weak cytotoxic effects. Molecular docking studies were performed to gain insights into the antibacterial activity mechanism of the synthesized compounds. The interactions and the orientation of compound 10a (1,1'-((disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-phenylurea)) were found to be highly similar to the original ligand within the binding pocket E. faecalis β-ketoacyl acyl carrier protein synthase III (FabH). Finally, a theoretical study was established to predict the physicochemical properties of the compounds.

Engineering Streptomyces coelicolor for production of monomethyl branched chain fatty acids

Branched chain fatty acids (BCFA) are an appealing biorefinery-driven target of fatty acid (FA) production. BCFAs typically have lower melting points compared to straight chain FAs, making them useful in lubricants and biofuels. Actinobacteria, especially Streptomyces species, have unique secondary metabolism that are capable of producing not only antibiotics, but also high percentage of BCFAs in their membrane lipids. Since biosynthesis of polyketide (PK) and FA partially share common pathways to generate acyl-CoA precursors, in theory, Streptomyces sp. with high levels of PK antibiotics production can be easily manipulated into strains producing high levels of BCFAs. To increase the percentage of the BCFA moieties in lipids, we redirected acyl-CoA precursor fluxes from PK into BCFAs using S. coelicolor M1146 (M1146) as a host strain. In addition, 3-ketoacyl acyl carrier protein synthase III and branched chain α-keto acid dehydrogenase were overexpressed to push fluxes of branched chain acyl-CoA precursors towards FA synthesis. The maximum titer of 354.1 mg/L BCFAs, 90.3% of the total FA moieties, was achieved using M1146dD-B, fadD deletion and bkdABC overexpression mutant of M1146 strain. Cell specific yield of 64.4 mg/L/gcell was also achieved. The production titer and specific yield are the highest ever reported in bacterial cells, which provides useful insights to develop an efficient host strain for BCFAs.