ATP synthase

Conidiospore germlings of Neuvospova crassa submitted to a heat shock at 4S'C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30°C). These observations suggest a temperaturedependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45'C while phosphorylase was activated. The reverse was true when the cells were shifted back to 3O'C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trebalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50°C than at 3O'C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30°C but not at 5072. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45°C or that of glycogen at 30°C.

Conidiospore germlings of Neuvospova crassa submitted to a heat shock at 4S'C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30°C). These observations suggest a temperaturedependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45'C while phosphorylase was activated. The reverse was true when the cells were shifted back to 3O'C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trebalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50°C than at 3O'C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30°C but not at 5072. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45°C or that of glycogen at 30°C.

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The K m and V max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min −1 μg −1 , respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

Estrogens are known to modulate lower urinary tract (LUT) trophicity and neuronal nitric oxide synthase (nNOS) expression in several organs. The aim of this study was to explore the effects of endogenous and supraestrus levels of 17β-estradiol (E2) on LUT and urethral nNOS expression and function. LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration, or castration plus chronic E2 supplementation (80 μg·kg−1·day−1, i.e., pregnancy level). The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher than those of ovariectomized mice. Urethral resistance was increased twofold in E2-treated mice, with no structural changes in urethra, supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive ...

Solanesol is a terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum). The present study aimed to investigate the regulation of solanesol accumulation in tobacco leaves induced by moderately high temperature (MHT). Exposure to MHT resulted in a significant increase in solanesol content, dry weight, and net photosynthetic rate in tobacco leaves. In MHT-exposed tobacco leaves, 492 and 1440 genes were significantly up- and downregulated, respectively, as revealed by RNA-sequencing. Functional enrichment analysis revealed that most of the differentially expressed genes (DEGs) were mainly related to secondary metabolite biosynthesis, metabolic pathway, carbohydrate metabolism, lipid metabolism, hydrolase activity, catalytic activity, and oxidation-reduction process. Moreover, 122 transcription factors of DEGs were divided into 22 families. Significant upregulation of N. tabacum 3-hydroxy-3-methylglutaryl-CoA r...

Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed t...

Although some of the most relevant elements contributing to uncertainty can potentially be evaluated in clinical laboratories, the effort required to undertake such an endeavor might be so great that it will be difficult to bring into general use the uncertainty of patients' results.

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3b-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1 H NMR and CD spectroscopies as (À)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional nonspecific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.

Flavone synthase I, a soluble 2-oxoglutarate-dependent dioxygenase catalyzing the oxidation of flavanones to flavones in several Apiaceae species, was induced in parsley cell cultures by continuous irradiation with ultraviolet/blue light for 20 h. The enzyme was extracted from these cells and purified by a revised purification protocol including the fractionation on hydroxyapatite, Fractogel EMD DEAE, and Mono Q anion exchangers, which resulted in an apparently homogeneous flavone synthase at approximately 10fold higher yield as compared to the previous report. The homogeneous enzyme was employed to raise an antiserum in rabbit for partial immunological characterization. The specificity of the polyclonal antibodies was demonstrated by immunotitration and Western blotting of the crude ammonium sulfate-fractionated enzyme as well as of the enzyme at various stages of the purification. High titer cross-reactivity was observed toward flavone synthase I, showing two bands in the crude extract corresponding to molecular weights of 44 and 41 kDa, respectively, while only the 41 kDa was detected on further purification. The polyclonal antiserum did not cross-react with recombinantly expressed flavanone 3␤-hydroxylase from Petunia hybrida or flavonol synthase from Citrus unshiu, two related 2-oxoglutarate-dependent dioxygenases involved in the flavonoid pathway.

Cellulose, an abundant, crystalline polysaccharide, is central to plant morphogenesis and to many industries. Chemical and ultrastructural analyses together with map-based cloning indicate that the RSW1 locus of Arabidopsis encodes the catalytic subunit of cellulose synthase. The cloned gene complements the rsw1 mutant whose temperature-sensitive allele is changed in one amino acid. The mutant allele causes a specific reduction in cellulose synthesis, accumulation of noncrystalline β-1,4-glucan, disassembly of cellulose synthase, and widespread morphological abnormalities. Microfibril crystallization may require proper assembly of the RSW1 gene product into synthase complexes whereas glucan biosynthesis per se does not.

The hydride transfer from C6 of tetrahydrofolate to the reaction's exocyclic methylene-dUMP intermediate is the rate limiting step in thymidylate synthase (TSase) catalysis. This step has been studied by means of QM/MM molecular dynamics simulations to generate the corresponding free energy surfaces. The use of two different initial X-ray structures has allowed exploring different conformational spaces and the existence of chemical paths with not only different reactivities but also different reaction mechanisms. The results confirm that this chemical conversion takes place preferentially via a concerted mechanism where the hydride transfer is conjugated to thiol-elimination from the product. The findings also confirm the labile character of the substrate-enzyme covalent bond established between the C6 of the nucleotide substrate and a conserved cysteine residue. The calculations also reproduce and rationalize a normal H/T 2° kinetic isotope effect measured for that step. From a...

The reaction of ATP synthesis obeys the general thermodynamic relation between the rate constants for the forward and backward reactions: the ratio of these constants, i.e. the equilibrium constant depends only on the difference of the electrochemical potentials between two sides of the membrane ∆µ ~Н+, or proton motive force ∆µ ~Н+/F (pmf), and is independent of its individual components, the gradients of the electrical ∆ϕ = ϕ′ -ϕ′′ and chemical ∆µ Н + potentials (EP and CP). However, each of the two rates can differently depend on the pmf components, which manifests under non-equilibrium conditions created by the work of the proton pumps. The question of the actual form of this dependence is still debated: some authors believe that there is no such dependence [1, 2], and others suggest that there is a dependence upon pH [3]. Here we consider

Mitochondria cannot maximize energy production, efficiency, and the cellular ATP phosphorylation potential all at the same time. The theoretical and observed determinations of coupling of oxidative phosphorylation in mitochondria from rat liver, heart, and brain were compared using classical and nonequilibrium thermodynamic measures. Additionally, the optimal thermodynamic efficiency and flow ratios were determined for control of the two energy-converting complexes of the respiratory chain: complex I (NADH), which reflects the integrated cellular pathway, and complex II (FADH2), the predominantly tricarboxylic acid (TCA) cycle pathway. For all three organs, the cellular respiratory pathway was more tightly coupled than the TCA pathway and resulted in a greater optimal efficiency. Liver mitochondria are the most thermodynamically efficient at ATP production using oxidative phosphorylation. Heart and brain mitochondrial systems utilize more oxygen, but can produce ATP at a faster rate...

Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

Biosynthesis of vitamin B6 is essential for all living cells. Most organisms use the pyridoxal 5’-phosphate (PLP) synthase complex to synthesize the cofactor form, PLP, from the three substrates ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P) and ammonia. PLP synthase complex is a glutamine amidotransferase (GATase) class I, consisting of 12 Pdx1 and 12 Pdx2 subunits. Pdx1 is responsible for the PLP synthesis and Pdx2 is the glutaminase that hydrolyses glutamine to produce ammonia, which is transfered to the Pdx1 active site. In this PhD Thesis, studying Pdx1 and Pdx2 proteins from the human parasite Plasmodium falciparum and from the rodent parasite Plasmodium berghei gave important insights into the assembly, activation and substrate tunneling of the PLP synthase complex. Electron microscopy analyses showed that association of the PLP synthase and glutaminase subunits was random, suggesting a non cooperative mechanism independent of neighboring Pdx1 binding sites to be ...

INTRODUCTION. PAPS is the universal sulfonate donor compound. PAPS synthase (PAPSS) catalyzes the formation PAPS in two concerted steps. In the first step SO 4 -combines with ATP to form adenosine 5'-phosphosulfate (APS) and PPi catalyzed by ATP sulfurylase domain. In the second step APS combines with another molecule of ATP to form PAPS and ADP catalyzed by APS kinase domain. In higher eukaryotes APS kinase domain (aa 1-268) and ATP sulfurylase domain (aa 220-623) are fused together into a single polypeptide termed PAPS synthase (PAPSS) (1). cDNA of the two isoforms PAPSS1 (accession # U53447) and PAPSS2b (accession # AF 150754) from human have been molecularly cloned and characterized[1]. PAPS synthase genes have been localized to chromosome 4q24 (PAPSS1) and chromosome 10q22.3-10q25.1 (PAPSS2)[1,2].

The mite Sarcoptes scabiei causes sarcoptic mange (or scabies), a disease of considerable human and veterinary significance. An S. scabiei cDNA clone of about 2 kb was isolated from a S. scabiei var. hominis expression library by immunological screening using blood serum from a naturally infected chamois (Rupicapra rupicapra). The nucleotide sequence of the identified cDNA contains an open reading frame of 1930 bp that encodes a 642 amino acid polypeptide. This polypeptide shows tandem repeats of a glycine-serine rich 20 residue sequence followed by a unique C-terminal glutamate rich 54 residue sequence. The cDNA or the deduced polypeptide did not show significant similarities to any of the sequences in the databases. A carboxyl-terminal fragment of this polypeptide (residues 380 to 642) was efficiently expressed in Escherichia coli as a fusion with Glutathione S-transferase and then was used to produce a specific antiserum. The antigen encoded by the cDNA was located at the integument of the mite's epidermis and the cavities surrounding its vital organs. Western blot analysis of mite extracts using the specific antiserum against the recombinant protein identified antigens larger that 60 kDa indicating that the isolated cDNA did not contain the full ORF. Moreover, we designed a diagnostic assay based on the carboxyl-terminal fragment of the antigen for the identification of infected animals. Sarcoptes scabiei / cDNA library / immunolocalisation / sarcoptic mange diagnosis / ELISA

The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

In this paper, we describe the effects of the expression of GM3 synthase at high levels in human ovarian carcinoma cells. Overexpression of GM3 synthase in A2780 cells consistently resulted in elevated ganglioside (GM3, GM2 and GD1a) levels. GM3 synthase overexpressing cells had a growth rate similar to wild-type cells, but showed a strongly reduced in vitro cell motility accompanied by reduced levels of the epithelial-mesenchymal transition marker α smooth muscle actin. A similar reduction in cell motility was observed upon treatment with exogenous GM3, GM2, and GM1, but not with GD1a. A photolabeling experiment using radioactive and photoactivable GM3 highlighted several proteins directly interacting with GM3. Among those, caveolin-1 was identified as a GM3-interacting protein in GM3 synthase overexpressing cells. Remarkably, caveolin-1 was markedly upregulated in GM3 synthase overexpressing cells. In addition, the motility of low GM3 synthase expressing cells was also reduced in the presence of a Src kinase inhibitor; on the other hand, higher levels of the inactive form of c-Src were detected in GM3 synthase overexpressing cells, associated with a ganglioside-and caveolin-rich detergent insoluble fraction.

Threonine synthase, which is a PLP-dependent enzyme, catalyzes the ␤,␥-replacement reaction of L-homoserine phosphate to yield threonine and inorganic phosphate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its unliganded form and complexed with the substrate analogue 2-amino-5phosphonopentanoic acid have been determined at 2.15 and 2.0 Å resolution, respectively. The complexed form, assigned as an enamine, uncovered the interactions of the cofactor-analogue conjugate with the active site residues. The binding of the substrate analogue induces a large conformational change at the domain level. The small domain rotates by about 25°and approaches the large domain to close the active site. The complicated catalytic process of the enzyme has been elucidated based on the complex structure to reveal the stereochemistry of the reaction and to present the released inorganic phosphate as a possible catalyst to carry a proton to the C␥ atom of the substrate.

environmental contaminants. Here we present the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1. This is the first crystal structure of the biphenyl dioxygenase. We also determined the crystal structure of the BphA1A2-biphenyl complex. Structural comparison between the substrate free and complex forms revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. The analysis with the two crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which respectively seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket.

Several oncogenic proteins and tumour suppressors target the RNA polymerase I and interfere with rRNA synthesis. Here, we show that the glycogen synthase kinase (GSK) 3b, which phosphorylates the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), is selectively enriched in nucleoli of RAStransformed cells. Immunoprecipitation and chromatin immunoprecipitation assays performed on epithelial and endothelial cells transformed with oncogenic RAS show that GSK3b and PTEN are part of the same complex and associate with promoter and coding region of the rDNA. An active GSK3b mutant abolished nucleolar BrUTP incorporation and associated with the member of the selectivity factor 1 complex TAF I 110. Finally, GSK3b inhibition upregulated 45S, 18S and 28S rRNA synthesis in RAS-transformed epithelial cells as revealed by semiquantitative real-time PCR and promoted cellular proliferation. Our results underscore a repressive function for GSK3b in rRNA biogenesis supporting its role as a tumour supressor.

Ramulosis (Colletotrichum gossypii South var. cephalosporioides Costa) is a fungal disease of cotton (Gossypium hirsutum L.) that causes damage to leaves, stems, and bolls by reducing fiber production; it can be controlled by chemical fungicides. Geraniol is a monoterpenoid produced by some aromatic plant species whose fungicidal properties have been widely reported. Geraniol synthase (GES; EC 3.1.7.11) is the precursor enzyme involved in the biosynthetic chain of geraniol. Geraniol synthase (ges) gene transcripts were prospected in 11 aromatic species with molecular and phytopathological tools to identify promising accessions for further use in in vitro and in vivo assays involving the control of cotton ramulosis. Mentha pulegium L. oil highly expressed ges and inhibited fungal growth at 1000 μL L -1 in an in vitro assay. Validation assays were carried out in two environments and M. pulegium at 2000 μL L -1 reduced the initial and final severity indices of the disease to 48% and 52%, respectively, in preventive treatments; in curative assays, indices were 44% and 54%, respectively. This indicates that it is a promising bioactive compound to control cotton ramulosis.

Glycogen and starch are the major readily accessible energy storage compounds in nearly all living organisms. Glycogen is a very large branched glucose homopolymer containing about 90% a-1,4-glucosidic linkages and 10% a-1,6 linkages. Its synthesis and degradation constitute central pathways in the metabolism of living cells regulating a global carbon/energy buffer compartment. Glycogen biosynthesis involves the action of several enzymes among which glycogen synthase catalyzes the synthesis of the a-1,4-glucose backbone. We now report the first crystal structure of glycogen synthase in the presence and absence of adenosine diphosphate. The overall fold and the active site architecture of the protein are remarkably similar to those of glycogen phosphorylase, indicating a common catalytic mechanism and comparable substratebinding properties. In contrast to glycogen phosphorylase, glycogen synthase has a much wider catalytic cleft, which is predicted to undergo an important interdomain 'closure' movement during the catalytic cycle. The structures also provide useful hints to shed light on the allosteric regulation mechanisms of yeast/mammalian glycogen synthases.

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSerresistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mt-rRNA, with defects in the biogenesis of the mitoribosome large subunit (mt-LSU), and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA,...

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase ␤-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic ␤-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase ␤-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase ␤-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.

In higher plants, cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex. The catalytic core of the complex is believed to be composed of three types of CESA subunits. Indirect evidence suggests that the complex associated with primary wall cellulose deposition consists of CESA1, -3, and -6 in Arabidopsis thaliana . However, phenotypes associated with mutations in two of these genes, CESA1 and -6 , suggest unequal contribution by the different CESAs to overall enzymatic activity of the complex. We present evidence that the primary complex requires three unique types of components, CESA1-, CESA3-, and CESA6-related, for activity. Removal of any of these components results in gametophytic lethality due to pollen defects, demonstrating that primary-wall cellulose synthesis is necessary for pollen development. We also show that the CESA6-related CESAs are partially functionally redundant.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O 2 , and two electrons to prostaglandin endoperoxide H 2 (PGH 2 ). Formation of PGH 2 involves an initial oxygenation of arachidonate to yield PGG 2 catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG 2 to form PGH 2 catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co 3؉ -heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG 2 versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

Background/Aims: L5, the most negatively charged species of low-density lipoprotein (LDL), has been implicated in atherogenesis by inducing apoptosis of endothelial cells (ECs) and inhibiting the differentiation of endothelial progenitor cells. In this study, we compared the effects of LDL charge on cellular stress pathways leading to atherogenesis. Methods: We isolated L5 and L1 (the least negatively charged LDL) from the plasma of patients with familial hypercholesterolemia and used JC-1 staining to examine the effects of L5 and L1 on the mitochondrial membrane potential (DCm) in human umbilical vein ECs (HUVECs). Additionally, we characterized the gene expression profiles of 7 proteins involved in various types of cellular stress. Results: The DCm was severely compromised in HUVECs treated with L5. Furthermore, compared with L1, L5 induced a decrease in mRNA and protein expression of the endoplasmic reticulum (ER) chaperone proteins ORP150, Grp94, and Grp58, mitochondrial protein...

Plants use two distinct isoprenoid biosynthesis pathways: the methylerythritol phosphate (MEP) pathway and the mevalonic acid (MVA) pathway. 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) are the rate-limiting enzymes in the MEP pathway, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key regulatory enzyme in the MVA pathway. Previously, overexpression of Populus trichocarpa PtDXR in Nanlin 895 (Populus × euramericana cv. 'Nanlin 895') poplar was found to upregulate resistance to salt and drought stresses, and the transgenic poplars showed improved growth. In the present study, PtHMGR overexpressors (OEs) exhibited higher expression levels of DXS, DXR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) and lower expression levels of 2-C-methyl-d-erythritol4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) than non-transgenic poplars (NT). However, the poplar PtDXR-OEs showed upregulated expression levels of MEP-related genes and downregulated expression of MVA-related genes. Moreover, overexpression of PtDXR and PtHMGR in poplars caused changes in MVA-derived trans-zeatin-riboside (TZR), isopentenyl adenosine (IPA), castasterone (CS) and 6-deoxocastasterone (DCS), as well as MEP-derived .

Full wwPDB X-ray Structure Validation Report 1OFR 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: The reported resolution of this entry is 2.70 Å. Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) Clashscore 122078 2755 (2.70-2.70) Ramachandran outliers 120005 2715 (2.70-2.70) Sidechain outliers 119972 2715 (2.70-2.70) Full wwPDB X-ray Structure Validation Report 1OFR Continued from previous page... Mol Chain Length Quality of chain 1 H 370 Full wwPDB X-ray Structure Validation Report 1OFR 3 Residue-property plots i ○ These plots are drawn for all protein, RNA and DNA chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometryand electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey. Note EDS was not executed.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis. Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP-synthesis. Through medicinal chemistry exploration, we established a robust structure activity relationship of these two scaffolds resulting in nanomolar potencies in an ATP-synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be non-cytotoxic and demonstrated efficacy in a mouse model of tuberculosis...

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. FoF1-ATP synthase, a complex V of electron transport chain, is an important constituent of mitochondria dependent-signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3'-Diindolylmethane (DIM), a DNA topoisomerase I poison inhibits mitochondrial FoF1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of FoF1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial ROS production, followed by depolarization of mitochondrial membrane potential (ΔΨm). Since, ΔΨm is the driving force for mitochondrial ATP synthesis, loss of ΔΨm results in depletion of cellular ATP level. The loss of ΔΨm causes the cellular ROS generation and in turn led to the oxidative DNA lesions followed by DNA fragmentation. On the other hand, loss of ΔΨm leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mt-DNA depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our studies indicate for the first time that DIMinduced mitochondrial dysfunction by inhibition of FoF1-ATP synthase activity leads to PCD in Leishmania parasites, which could be exploited to develop newer potential therapeutic targets.

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F H F I and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F H F I . The proton transfer through F H F I after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATPsynthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

F O F 1 -ATP synthase converts two energetic bcurrenciesQ of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/ generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the singleenzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (b1 AM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of c0.25 mol ADP per mole of enzyme. There was no detectable detachment of F 1 from F O . (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (N1 AM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s À1 at pH 8. Proton slip probably presents the free-wheeling of the central rotary shaft, subunit g, in an open structure of the (ah) 3 hexagon with no nucleotides in the catalytic sites.

We detected a first case of 6-Pyruvoyl-tetrahydropterin synthase deficiency in Neuropediatric department mongi Ben Hmida of Tunisia. Genetic analyses of PTS gene demonstrated a homozygous mutation; treatment had been started at the age of 3 years.

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-"%C]acetyl-CoA-labelled Cys""", and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys"""Ser mutant, however, still Abbreviations used : KAS, 3-oxoacyl-(acyl-carrier-protein) synthase ; ACP, acyl carrier protein ; FAS, fatty acid synthase. 1 To whom correspondence should be addressed (e-mail spener!uni-muenster.de). was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His#'" with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His#'". Double mutants Cys"""Ser\His#'"Ala and Cys"""Ser\His#'"Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-"%C]malonyl-ACP, a reaction indicating the role of His#'" in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg"&! and Arg$!' in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

Background: Tissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. One of the compensatory mechanisms to alleviate the sepsis-induced mitochondrial dysfunction could be the increase in oxidative phosphorylation efficiency (ATP/O). We propose to study liver mitochondrial function and oxidative stress and the regulatory mechanism of mitochondrial oxidative phosphorylation efficiency in an animal model of sepsis. Methods: We induced sepsis in rats by cecal ligation and perforation (CLP). Six, 24, or 36 h following CLP, we measured liver mitochondrial respiration, cytochrome c oxidase activity, and membrane permeability. We determine oxidative phosphorylation efficiency, by measuring ATP synthesis related to oxygen consumption at various exogenous ADP concentrations. Finally, we measured radical oxygen species (ROS) generation by liver mitochondria and mRNA concentrations of UCP2, biogenesis factors, and cytokines at the same end points. Results: CLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit β content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression. Conclusions: Despite a compensatory increase in mitochondrial biogenesis factors, liver mitochondrial functions remain altered after CLP. This suggests that the functional compensatory mechanisms reported in the present study (slip at cytochrome c oxidase and biogenesis factors) were not strong enough to increase oxidative phosphorylation efficiency and failed to limit liver mitochondrial ROS over-generation. These data suggest that treatments based on cytochrome c infusion could have a role in mitochondrial dysfunction and/or ROS generation associated with sepsis.

The catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase from Escherichia coli was investigated under pre-steady-state conditions using rapid chemical quench flow methods. The results suggest the formation of acyclic bisphosphate 1 as a reaction intermediate.