The genome of oil-Camellia and population genomics analysis provide insights into seed oil domestication

Background As a perennial crop, oil-Camellia possesses a long domestication history and produces high-quality seed oil that is beneficial to human health. Camellia oleifera Abel. is a sister species to the tea plant, which is extensively cultivated for edible oil production. However, the molecular mechanism of the domestication of oil-Camellia is still limited due to the lack of sufficient genomic information. Results To elucidate the genetic and genomic basis of evolution and domestication, here we report a chromosome-scale reference genome of wild oil-Camellia (2.95 Gb), together with transcriptome sequencing data of 221 cultivars. The oil-Camellia genome, assembled by an integrative approach of multiple sequencing technologies, consists of a large proportion of repetitive elements (76.1%) and high heterozygosity (2.52%). We construct a genetic map of high-density corrected markers by sequencing the controlled-pollination hybrids. Genome-wide association studies reveal a subset of artificially selected genes that are involved in the oil biosynthesis and phytohormone pathways. Particularly, we identify the elite alleles of genes encoding sugar-dependent triacylglycerol lipase 1, β-ketoacyl-acyl carrier protein synthase III, and stearoyl-acyl carrier protein desaturases; these alleles play important roles in enhancing the yield and quality of seed oil during oil-Camellia domestication. Conclusions We generate a chromosome-scale reference genome for oil-Camellia plants and demonstrate that the artificial selection of elite alleles of genes involved in oil biosynthesis contributes to oil-Camellia domestication.

Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl-enzyme intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E. coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Binding site comparison between PaFabB and PaFabF revealed a conserved malonyl binding site but differences in the fatty acid binding channel. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.

GC-MS analysis and Molecular docking of Quercetin compounds of Phytolacca octandra with the target protein of infection causing Staphylococcus aureus

Phytolacca octandra is a perennial usually about 1m high herb, dense and erect in full sun. As only few reports were available on the studies about the bioactive compounds and various activities in the Phytolacca octandra, the present study focuses on the bio active compounds attributed to antibacterial activity in the plant extracts by Gas Chromatography – Mass Spectrometry (GCMS) and molecular docking methods. Antibacterial activity of Phytolacca octandra showed maximum inhibitory zones of 21mm, 18mm, 19mm, 19mm and 20mm against respective organisms for 25mg/ml of acetone extracts. The outcome of Phytolacca octandra extracts that was exposed to GC-MS analysis, showed the presence of 20 more compounds. The most identified compounds to have anti-oxidant activity are Dodecane, Octadecane and Octacosane. The other major compounds present in extract are Cyclohexen-oxopropyl, 1,2-Benzenedicarboxylic acid.The overall docking energies of the target protein, rhamnolipids biosynthesis 3-oxoacyl-[acyl-carrier-protein] reductase with quercetin with the number of hydrogen bonds were presented in the study; The docking report revealed –8.01Kcal/Mol binding energies and 8 hydrogen bonding between the Phytolacca octandra compound, quercetin and the target binding protein, rhlG of infection causing pathogen Staphylococcus aureus.

Structural stability of Cutibacterium acnes acyl carrier protein studied using CD and NMR spectroscopy

To survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur containing cofactor, indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by Lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential action of Lipoate protein ligase B (LipB) and Lipoyl synthase (LipA). LipB uptakes octanoyl- chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. The molecular basis of substrate recognition by LipB is still not fully understood. Using E. coli LipB as a model enzyme, we show that an octanoyl-transferase mainly recognizes the 4-phosphopantetheine tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. LipB can accept octanoate- from its own ACP, noncognate ACPs, as well as C8-CoA. Further, our NMR studies demonstrate the presence of an adenine and phosphate binding site in LipB, akin to LplA. A loop containing 71RGG73 sequence, analogous to the lipoate binding loop of LplA is also conserved in LipB. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge might be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

In-silico Studies of Thiopyridine Compounds as Anti-Bacterial agents Targeting Enoyl - Acyl Carrier Protein Reductase

In the Fatty Acid Synthase II system, Enoyl-(acyl-carrier-protein) reductase (ENR) encoded by FabI genes is a limiting step enzyme and there is no homologue ENR found in invertebrates which makes it selective target for drug discovery. From Molecular dynamics simulations it was concluded that the solvated protein stabilized at 2.5 ns with larger mobility in the substrate - binding loop and the conformational flexibility of the molecule was revealed. To study the inhibitory effects of novel small molecules in the thiopyridine series, a 2D QSAR model was developed and evaluated for its efficiency. The R2 > 0.96 and Q2 = 0.978 depicted the predictive ability of the models which was determined using a test set of 3 compounds. The receptor-ligand interactions were studied and highest affinity was reported for GCT ID, 343129 (-9.09 Kcal/mol), 341772 (-8.90 Kcal/mol) and 268776 (-8.85 Kcal/mol). These compounds were analysed for their drug like properties and toxicity which projected acceptable blood brain barrier permeation and human intestinal absorption and reduced lipotoxicity. Thus the results suggest further synthesis of new thipyridine series of compounds and experimental testing against drug resistant Staphylococcal infections

Combined Transcriptome and Lipidomic Analyses of Lipid Biosynthesis in Macadamia ternifolia Nuts

Macadamia nuts are considered a high-quality oil crop worldwide. To date, the lipid diversity and the genetic factors that mediate storage lipid biosynthesis in Macadamia ternifolia are poorly known. Here, we performed a comprehensive transcriptomic and lipidomic data analysis to understand the mechanism of lipid biosynthesis by using young, medium-aged, and mature fruit kernels. Our lipidomic analysis showed that the M. ternifolia kernel was a rich source of unsaturated fatty acids. Moreover, different species of triacylglycerols, diacylglycerol, ceramides, phosphatidylethanolamine, and phosphatidic acid had altered accumulations during the developmental stages. The transcriptome analysis revealed a large percentage of differently expressed genes during the different stages of macadamia growth. Most of the genes with significant differential expression performed functional activity of oxidoreductase and were enriched in the secondary metabolite pathway. The integration of lipidomic and transcriptomic data allowed for the identification of glycerol-3-phosphate acyltransferase, diacylglycerol kinase, phosphatidylinositols, nonspecific phospholipase C, pyruvate kinase 2, 3-ketoacyl-acyl carrier protein reductase, and linoleate 9S-lipoxygenase as putative candidate genes involved in lipid biosynthesis, storage, and oil quality. Our study found comprehensive datasets of lipidomic and transcriptomic changes in the developing kernel of M. ternifolia. In addition, the identification of candidate genes provides essential prerequisites to understand the molecular mechanism of lipid biosynthesis in the kernel of M. ternifolia.

Subtractive genomics and molecular docking approach to identify drug targets against Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a multidrug resistant pathogen associated with high mortality and morbidity in patients having compromised immunity. The efflux systems of S. maltophilia include SmeABC and SmeDEF proteins, which assist in acquisition of multiple-drug-resistance. In this study, proteome based mapping was utilized to find out the potential drug targets for S. maltophilia strain k279a. Various tools of computational biology were applied to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. The CD-HIT analysis selected 4315 proteins from total proteome count of 4365 proteins. Geptop identified 407 essential proteins, while the BlastP revealed approximately 85 non-homologous proteins in the human genome. Moreover, metabolic pathway and subcellular location analysis were performed for essential bacterial genes, to describe their role in various cellular processes. Only two essential proteins (Acyl-[acyl-carrier-protein]—UDP-N acetyl glucosamine O-acyltransferase and D-alanine-D-alanine ligase) as candidate for potent targets were found in proteome of the pathogen, in order to design new drugs. An online tool, Swiss model was employed to model the 3D structures of both target proteins. A library of 5000 phytochemicals was docked against those proteins through the molecular operating environment (MOE). That resulted in to eight inhibitors for both proteins i.e. enterodiol, aloin, ononin and rhinacanthinF for the Acyl-[acyl-carrier-protein]—UDP-N acetyl glucosamine O-acyltransferase, and rhazin, alkannin beta, aloesin and ancistrocladine for the D-alanine-D-alanine ligase. Finally the ADMET was done through ADMETsar. This study supported the development of natural as well as cost-effective drugs against S. maltophilia. These inhibitors displayed the effective binding interactions and safe drug profiles. However, further in vivo and in vitro validation experiment might be performed to check their drug effectiveness, biocompatibility and their role as effective inhibitors.

Design and Synthesis of Highly Active Antimycobacterial Mutual Esters of 2-(2-Isonicotinoylhydrazineylidene)propanoic Acid

The combination of two active scaffolds into one molecule represents a proven approach in drug design to overcome microbial drug resistance. We designed and synthesized more lipophilic esters of 2-(2-isonicotinoylhydrazineylidene)propanoic acid, obtained from antitubercular drug isoniazid, with various alcohols, phenols and thiols, including several drugs, using carbodiimide-mediated coupling. Nineteen new esters were evaluated as potential antimycobacterial agents against drug-sensitive Mycobacterium tuberculosis (Mtb.) H37Rv, Mycobacterium avium and Mycobacterium kansasii. Selected derivatives were also tested for inhibition of multidrug-resistant (MDR) Mtb., and their mechanism of action was investigated. The esters exhibited high activity against Mtb. (minimum inhibitory concentrations, MIC, from ≤0.125 μM), M. kansasii, M. avium as well as MDR strains (MIC from 0.25, 32 and 8 µM, respectively). The most active mutual derivatives were derived from 4-chloro/phenoxy-phenols, triclosan, quinolin-8-ol, naphthols and terpene alcohols. The experiments identified enoyl-acyl carrier protein reductase (InhA), and thus mycobacterial cell wall biosynthesis, as the main target of the molecules that are activated by KatG, but for some compounds can also be expected adjunctive mechanism(s). Generally, the mutual esters have also avoided cytotoxicity and are promising hits for the discovery of antimycobacterial drugs with improved properties compared to parent isoniazid.

Bacterial Product Template Domain: An Evolutionary Intermediate between Dehydratase and Aldol Cyclase of Type I Polyketide Synthases

The product template (PT) domains act as an aldol cyclase to control the regiospecific aldol cyclization of the extremely reactive poly-β-ketone intermediate assembled by an iterative type I polyketide synthases (PKSs). Up to now, only the structure of fungal PksA PT that mediates the first-ring cyclization via C4-C9 aldol cyclization is available. We describe here the structural and computational characterization of a bacteria PT domain that controls C2-C7 cyclization in orsellinic acid (OSA) synthesis. Mutating the catalytic His949 of the PT abolishes production of OSA and results in a tetraacetic acid lactone (TTL) generated by spontaneous O-C cyclization of the acyl carrier protein (ACP)-bound tetraketide intermediate. Crystal structure of the bacterial PT domain closely resembles dehydrase (DH) domains of modular type I PKSs in the overall fold, dimerization interface and catalytic “His-Asp” dyad organization, but is significantly different from PTs of fungal iterative type I PKSs. QM/MM calculation reveals that the catalytic His949 abstracts a proton from C2 and transfers it to C7 carbonyl to mediate the cyclization reaction. According to the structural similarity to DHs and the functional similarity to fungal PTs, we propose that the bacterial PT represents an evolutionary intermediate between the two tailoring domains of type I PKSs.