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1.
β-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes the initial step of fatty acid biosynthesis via a type II fatty acid synthase in most bacteria. The important role of this essential enzyme combined with its unique structural features and ubiquitous occurrence in bacteria has made it an attractive new target for the development of new FabH inhibitors. We first used a structure-based approach to develop 24 new vinylogous carbamates (4a-15a, 4b-15b) that target FabH for the development of new antibiotics in this paper. Potent FabH inhibitory and selective anti- Gram-negative bacteria activities were observed in most of these vinylogous carbamates. Especially, compound 6a and 7a showed the most potent FabH inhibitory activity with IC?? of 2.6 and 3.3 μM, respectively. Docking simulation was performed to position compound 6a into the Escherichia coli FabH active site and the possible binding conformation of compounds has been proposed. The biological data and molecular docking indicated that compounds 6a and 7a were potent inhibitors of E. coli FabH as antibiotics deserving further research. 相似文献
2.
Damián González-Mellado Penny von Wettstein-Knowles Rafael Garcés Enrique Martínez-Force 《Planta》2010,231(6):1277-1289
The β-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step
of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids
in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like
ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that
HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds
storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized
plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding
C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains. 相似文献
3.
The cerulenin-insensitive -ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C4,C6,C8 and C10) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 M cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS III-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC50 = 0.45 M), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata seeds by decanoyl-ACP was approximately 1.5-fold higher. The data provide evidence that acyl-ACPs are involved in the modulation of plant fatty-acid biosynthesis by a feed-back mechanism.Abbreviations ACP
acyl carrier protein
- DTT
dithiothreitol
- TCA
trichloroacetic acid
- ecACP
acyl carrier protein from Escherichia coli
- FAS
fatty-acid synthase
- IC50
concentration causing 50% inhibition
- KAS
-ketoacyl-ACP synthase
- NEM
N-ethylmaleimide
In honour of Professor Hartmut K. Lichtenthaler's sixtieth birthdayThis work was supported by a grant from the German Ministry of Research and Technology (BMFT) and in part by the Fonds der Chemischen Industrie and the Ministry of Science and Research of the State Northrhine-Westfalia. The authors wish to thank Prof. G. Röbbelen (University of Göttingen, Göttingen, Germany) for kindly providing the plant material. This paper is part of the doctoral thesis of Fritzi Maike Brück. 相似文献
4.
A series of novel cinnamic acid secnidazole ester derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Compound 3n showed the most potent antibacterial activity with MIC of 1.56-6.25 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC?? of 2.5 μM. Docking simulation was performed to position compound 3n into the E. coli FabH active site to determine the probable binding conformation. 相似文献
5.
6.
Yang YS Zhang F Gao C Zhang YB Wang XL Tang JF Sun J Gong HB Zhu HL 《Bioorganic & medicinal chemistry letters》2012,22(14):4619-4624
A series of sulfur-containing heterocyclic pyrazoline derivatives (C1-C18; D1-D9) have been synthesized and purified (all are new except one) to be screened for FabH inhibitory activity. Compound C14 showed the most potent biological activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis (MIC values: 1.56-3.13 μg/mL), being comparable with the positive control, while D6 performed the best in the thiazolidinone series (MIC values: 3.13-6.25 μg/mL). They also demonstrated strong broad-spectrum antimicrobial activity. Compounds C14 and D6 exhibited the most potent E. coli FabH inhibitory activity with IC(50) of 4.6 and 8.4 μM, respectively, comparable with the positive control DDCP (IC(50)=2.8 μM). Docking simulation was performed to position compound C14 and D6 into the E. coli FabH structure active site to determine the probable binding model. The structurally modification of previous compounds and the attempt in innovative target have brought a positive progress. 相似文献
7.
We have cloned and sequenced the genes atpB and atpE, coding for CF1 subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F1/CF1- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae. 相似文献
8.
Immaculada Martin Josep A. Villena Marta Giralt Roser Iglesias Teresa Mampel Octavi Vińas Francesc Villarroya 《Molecular and cellular biochemistry》1996,154(2):107-111
The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T3 when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T3 in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms. 相似文献
9.
Lipid metabolism plays a pivotal role in cell structure and in multiple plant developmental processes. β-Ketoacyl-[acyl carrier protein] synthase I (KASI) catalyzes the elongation of de novo fatty acid (FA) synthesis. Here, we report the functional characterization of KASI in the regulation of chloroplast division and embryo development. Phenotypic observation of an Arabidopsis thaliana T-DNA insertion mutant, kasI, revealed multiple morphological defects, including chlorotic (in netted patches) and curly leaves, reduced fertility, and semidwarfism. There are only one to five enlarged chloroplasts in the mesophyll cells of chlorotic sectors of young kasI rosette leaves, indicating suppressed chloroplast division under KASI deficiency. KASI deficiency results in a significant change in the polar lipid composition, which causes the suppressed expression of FtsZ and Min system genes, disordered Z-ring placement in the oversized chloroplast, and inhibited polymerization of FtsZ protein at mid-site of the chloroplast in kasI. In addition, KASI deficiency results in disrupted embryo development before the globular stage and dramatically reduces FA levels (~33.6% of the wild type) in seeds. These results demonstrate that de novo FA synthesis is crucial and has pleiotropic effects on plant growth. The polar lipid supply is important for chloroplast division and development, revealing a key function of FA synthesis in plastid development. 相似文献
10.
Maity K Venkata BS Kapoor N Surolia N Surolia A Suguna K 《Journal of structural biology》2011,176(2):238-249
The β-hydroxyacyl-acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ is available in hexameric (active) and dimeric (inactive) forms. However, PfFabZ has not been crystallized with any bound inhibitors until now. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of four of these complexes. This is the first report on any FabZ enzyme with active site inhibitors that interact directly with the catalytic residues. Inhibitor binding not only stabilized the substrate binding loop but also revealed that the substrate binding tunnel has an overall shape of “U”. In the crystal structures, residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed. Thus, Phe169, merely by changing its side chain conformation, appears to be controlling the length of the tunnel to make it suitable for accommodating longer substrates. The volume of the substrate binding tunnel is determined by the sequence as well as by the conformation of the substrate binding loop region and varies between organisms for accommodating fatty acids of different chain lengths. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms. 相似文献
11.
12.
Milagros Balbín Magnus Abrahamson Lars Gustafson Karin Nilsson Arne Brun Anders Grubb 《Human genetics》1992,89(5):580-582
Summary A novel mutation, a C to T transition at base pair 2124 in exon 17 of the amyloid -protein precursor (APP) gene, has been identified by direct sequencing of amplified DNA from two Alzheimer's disease (AD) patients. A simple oligonucleotide-hybridization procedure was developed to allow population studies of this DNA variation. The mutation, which is silent at the protein level, was present in 2 out of 12 investigated AD patients, in 1 out of 60 non-AD patients and in 1 out of 30 healthy individuals. The mutation can be used as a new marker for linkage studies involving the APP gene, although more comprehensive population studies are required to determine the status of the mutation as a possible risk factor for the development of AD. 相似文献
13.
Characteristics of state II—state III transitions in the red alga, Porphyra perforata, were studied by measuring the fluorescence time course at room temperature and fluorescence spectra at 77 K. The state II to III transition was induced by system II light and was sensitive to uncouplers of photophosphorylation. This state II to III transition has a dark step(s) that could be easily separated from the light process. A state III to II transition occurred in the dark, but system I light accelerated the transition. The accelerating effect of system I light was not sensitive to uncouplers of photophosphorylation, but was inhibited by the addition of valinomycin + KCl or antimycin A. Compared to state I—state II transitions, the state II—state III transitions occurred more rapidly. The state II to state III transitions are different from the state I to state II transitions in that in state III the activity of photosystem II is changed without having any effect on photosystem I activity (Satoh and Fork, Biochim. Biophys, Acta, in press, 1982). It is suggested that the state II—state III transition represents a mechanism by which the alga can avoid photodamage resulting from absorption of excess light energy by photosystem II. 相似文献
14.
Kenneth H. Astrin Cecilia A. Warner Han-Wook Yoo Paul J. Goodfellow Shih-Feng Tsai Robert J. Desnick 《Human genetics》1991,87(1):18-22
Summary Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2 q26.3. 相似文献
15.
Cr(III), which is thought to be relatively non-toxic, was reduced to Cr(II) ion by biological reductants such as L-cysteine and NADH and Cr(II) thus formed could easily react with hydrogen peroxide (H2O2) to yield very reactive active oxygen species, hydroxyl radical (.OH). The formation of hydroxyl radical was detected by water-soluble spin-traps, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). This result indicates that non-toxic Cr(III) compounds have the possibility of causing dangerous effects to living organism in the presence of biological reductants. 相似文献
16.
Lijin Shu John A. Broadwater Catalina Achim Brian G. Fox Eckard Münck L. Que Jr 《Journal of biological inorganic chemistry》1998,3(4):392-400
Stearoyl-acyl carrier protein (ACP) Δ9-desaturase (Δ9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O2- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation state. These studies reveal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41?Å. The species having the 3.12?Å Fe-Fe distance also exhibits a 1.8?Å Fe-O bond and is thus proposed to represent Δ9D molecules containing a (μ-oxo)bis(μ-carboxylato)diiron(III) cluster. The species having the 3.41?Å Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Δ9D molecules containing a (μ-hydroxo)diiron(III) cluster. Mössbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (ΔE Q(1)=1.53?mm/s, 72%;ΔE Q(2)=0.72?mm/s, 21%;ΔE Q(3)=2.20?mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (μ-oxo)- and (μ-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mössbauer majority species, is likely associated with the EXAFS majority species having a 3.12?Å Fe-Fe separation and a 1.8?Å Fe-μ-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41?Å Fe-Fe separation. The presence of multiple diiron center conformations in diferric Δ9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (~9?kDa) during desaturation catalysis. 相似文献
17.
Pestov NB Zhao H Basrur V Modyanov NN 《Biochemical and biophysical research communications》2011,(4):543-548
ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS–PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9 kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions. 相似文献
18.
Partha Sen Caryl A. Chlan Mark D. Burow Woo Sung Lee Norimoto Murai 《Transgenic research》1993,2(1):21-28
A previous analysis with deletion mutants of the native -phaseolin gene demonstrated that removal of a negative element 5 upstream of–107 permitted phaseolin expression in stem cortex and secondary root (Burowet al., 1992). Here we employed the -glucuronidase (GUS) reporter gene to visualize, by histochemical staining, the cell type-specificity of phaseolin expression in stem and root, and to understand further the spatial control of the -phaseolin gene. The 782 bp 5 upstream promoter and its deletion mutants were fused to the GUS gene, and these chimaeric genes were used to transform tobacco. Histochemical staining for GUS activity demonstrated that phaseolin promoters truncated downstream of –227 conferred cell-type specific expression in internal/external phloem and protoxylem of mature stem. Surprisingly, GUS staining was prominent in both apical and lateral shoot apices of plants that contain the full-length –782 promoter and mutant promoters deleted up to –64. GUS expression was extended to all cell types of shoot tips, including epidermis, cortex, vasculature, procambium and pith. Expression in vasculature of petioles was limited to plants with promoters truncated to –106 and –64. The current results are in agreement with our previous findings with the native phaseolin gene: that the major positive element (–295/–228) is sufficient for seed-specific late-temporal expression of the phaseolin gene. We conclude that the 5 upstream sequence of the -phaseolin gene directs spatially- and temporally-controlled gene expression in developing seeds during the reproductive phase, but also confers expression in shoot apices during the vegetative phase of plant development. 相似文献
19.
George P. Patrinos Aphrodite Loutradi-Anagnostou Manoussos N. Papadakis 《Human genetics》1996,97(3):357-358
A new TA base substitution, identified inside the 5 regulatory region of the humanA globin gene (A –499 T A), is reported. This nucleotide change was found to be linked incis with the mutation producing sickle cell anemia (CD6 GAGGTG: s gene). 相似文献