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Toxoplasma gondii is an opportunistic obligate parasite, ubiquitous around the globe with seropositivity rates that range from 10% to 90% and infection by the parasite of pregnant women causes pre-natal death of the foetus in most cases and severe neurodegenerative syndromes in some. No vaccine is currently available, and since drug-resistance is common among T. gondii strains, discovering lead compounds for drug design using diverse tactics is necessary. In this study, the sole constituent isoform of an enzymatic 3-oxoacyl-[acyl-carrier-protein] reduction step in an apicoplast-located fatty acid biosynthesis pathway was chosen as a possible drug target. FASII is prokaryotic therefore, targeting it would pose fewer side-effects to human hosts. After a homology 3D modelling of TgFabG, a high-throughput virtual screening of 9867 compounds, the elimination of ligands was carried out by a flexible ligand molecular docking and 200 ns molecular dynamics simulations, with additional DCCM and PC plot analyses. Molecular Dynamics and related post-MD analyses of the top 3 TgFabG binders selected for optimal free binding energies, showed that L2 maintained strong H-bonds with TgFabG and facilitated structural reorientation expected of FabGs, namely an expansion of the Rossmann Fold and a flexible lid capping. The most flexible TgFabG sites were the α7 helix (the flexible lid region) and the β4-α4 and β5-α6 loops. For TgFabG-L2, the movements of these regions toward the active site enabled greater ligand stability. Thus, L2 ("Skimmine"; PubChem ID: 320361), was ultimately selected as the optimal candidate for the discovery of lead compounds for rational drug design.Communicated by Ramaswamy H. Sarma.

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis ( Arabidopsis thaliana ) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO 2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Ethyl ( S )-3-hydroxy-3-(2-thienyl)propanoate(( S )-HEES)acts as a key chiral intermediate for the blockbuster antidepressant drug duloxetine, which canbe achieved viathe stereoselective bioreduction ofethyl 3-oxo-3-(2-thienyl) propanoate (KEES) that containsa 3-oxoacyl structure.The sequences of the short-chain dehydrogenase/reductases from Chryseobacterium sp. CA49 were analyzed, and the putative3-oxoacyl-acyl-carrier-protein reductase, Ch KRED12, was able to stereoselectivelycatalyze theNADPH-dependent reduction to produce ( S )-HEES.The reductase activity of Ch KRED12 towardsothersubstrates with 3-oxoacyl structure were confirmed with excellent stereoselectivity (>99% enantiomeric excess) in most cases. When coupled with a cofactor recycling system using glucose dehydrogenase, the Ch KRED12 was able to catalyze the complete conversion of 100 g/l KEES within 12h, yielding the enantiopure product with >99% ee, showing a remarkable potential to produce ( S )-HEES.

Key Message: A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F 2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC-MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla - Laf - fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Unlabelled: Silver nanoparticles (Ag-NPs) are excessively used as antibacterial agents; however, environmental interaction specifically with the plants remain uncertain. To study the size-dependent effects of Ag-NPs on soybean under flooding, a proteomic technique was used. Morphological analysis revealed that treatment with Ag-NPs of 15nm promoted soybean growth under flooding compared to 2 and 50-80nm. A total of 228 common proteins that significantly changed in abundance under flooding without and with Ag-NPs of 2, 15, and 50-80nm. Under varying sizes of Ag-NPs, number of protein synthesis related proteins decreased compared to flooding while number of amino acid synthesis related proteins were increased under Ag-NPs of 15nm. Hierarchical clustering identified the ribosomal proteins that increased under Ag-NPs of 15nm while decreased under other sizes. In silico protein-protein interaction indicated the beta ketoacyl reducatse 1 as the most interacted protein under Ag-NPs of 15nm while least interacted under other sizes. The beta ketoacyl reductase 1 was up-regulated under Ag-NPs of 15nm while its enzyme activity was decreased. These results suggest that the different sizes of Ag-NPs might affect the soybean growth under flooding by regulating the proteins related to amino acid synthesis and wax formation., Biological Significance: This study highlighted the response of soybean proteins towards varying sizes of Ag NPs under flooding stress using gel-free proteomic technique. The Ag NPs of 15nm improved the length of root including hypocotyl of soybean. The proteins related to protein metabolism, cell division/organization, and amino acid metabolism were differentially changed under the varying sizes of Ag NPs. The protein synthesis-related proteins were decreased while amino acid metabolism-related proteins were increased under varying sizes of Ag NPs. The ribosomal proteins were increased under Ag NPs of 15nm. The beta ketoacyl reductase 1 was identified as the most interacted protein under varying sizes of Ag NPs. The mRNA expression level of beta ketoacyl reductase was up-regulated under Ag NPs of 15nm while its activity was decreased. These results suggest that the Ag NPs of 15nm improved the soybean growth under flooding stress by increasing the proteins related to amino acid synthesis and waxes formation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

Unlabelled: β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants., Importance: This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: FabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth., Methods: Complementation of Escherichia coli fabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains. , Results: We report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants., Conclusion: R. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes.

PhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrier-protein reductase) catalyzes the β-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a high-performance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Comamonas testosteroni (C. testosteroni) is a gram negative bacterium which can use steroid as a carbon source and degrade steroid with about 20 special enzymes. Most of the enzymes are inducible enzymes. 3-Oxoacyl-ACP reductase (E.C. 1.1.1.100) alternatively known as β-ketoacyl-ACP reductase (BKR) is involved in fatty acid syntheses. DNA sequence comparison showed that BKR belongs to the short-chain alcohol dehydrogenase (SDR) family. Our results showed that BKR is necessary for the degradation of steroid hormones in C. testosteroni. The DNA fragment of the BKR gene was cloned into an expressional plasmid pET-15b. BKR protein was expressed with 6× His-tag on the N-terminus and the enzyme was purified with Ni-column. Antibodies against BKR were prepared and a new BKR quantitative ELISA was created in our laboratory. The purified BKR is a 30.6 kDa protein on SDS-PAGE. C. testosteroni was induced by testosterone, estradiol, estriol and cholesterol. The expression of BKR was detected with an ELISA. The result showed that the BKR expression could be induced by cholesterol and estriol but not by testosterone and estradiol. BKR gene knock-out mutant (M-C.T.) was prepared by homologous integration. High performance liquid chromatography (HPLC) was used to detect steroid hormone degradation in C. testosteroni ATCC11996 and BKR gene knock-out mutant. We proved that the M-C.T. eliminated of testosterone degradation. Degradations of cholesterol and estradiol were also decreased. We conclude that the novel BKR in C. testosteroni plays an important role in steroid degradation. This work provides some new information of SDR and steroid degradation in C. testosteroni., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain β-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new mutation in mabA, Thr4Ile, was identified in a Mycobacterium tuberculosis isolate from a patient whose culture remained positive after treatment. The same mutation was found in another 5 patients infected by different strains. A putative role for this mutation in the process of diminishing susceptibility to isoniazid is evaluated., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus., (© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Background: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR., Methodology/principal Findings: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells., Conclusions/significance: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.