Small decreases in SBPase cause a linear decline in the apparent RuBP regeneration rate, but do not affect Rubisco carboxylation capacity.
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The response of net photosynthetic CO(2) uptake (A) to increasing leaf intercellular CO(2) concentration (c(i)) was determined in antisense Nicotiana tabacum plants, derived from six independent transformation lines, displaying a range of sedoheptulose-1, 7-bisphosphatase (SBPase) activities. The maximum in vivo ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V(c,max)) and RuBP regeneration (J(max)) rates were calculated from the steady-state measurements of the A to c(i) response curves. In plants with reductions in SBPase activity of between 9% and 60%, maximum RuBP regeneration capacity declined linearly (r(2)=0.79) and no significant change in apparent in vivo Rubisco activity (V(c,max)) was observed in these plants. No correlation between V(c,max) and a decrease in capacity for RuBP regeneration was observed (r(2)=0.14) in the SBPase antisense plants. These data demonstrate that small decreases in SBPase activity limit photosynthetic carbon assimilation by reducing the capacity for RuBP regeneration. (+info)
Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria.
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In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase. (+info)
Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.
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The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate. (+info)
Adenophostin A and ribophostin, but not inositol 1,4,5-trisphosphate or manno-adenophostin, activate the Ca2+ release-activated Ca2+ current, I(CRAC), in weak intracellular Ca2+ buffer.
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Under physiological conditions of weak intracellular Ca(2+) buffering (0.1 mM EGTA), the second messenger Ins(1,4,5)P(3) often fails to activate any detectable store-operated Ca(2+) current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P(3) for Ins(1,4,5)P(3) receptors] consistently activates the current under similar conditions. Here, whole-cell patch clamp experiments have been performed to examine how adenophostin A can activate the store-operated Ca(2+) current (I(CRAC)) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca(2+) buffer, saturating concentrations of adenophostin A activated I(CRAC) maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P(3). In a weak Ca(2+) buffer, adenophostin A consistently activated I(CRAC), but the current was submaximal. High concentrations of Ins(1,4,5)P(3) or the non-metabolizable analogue Ins(2,4,5)P(3) were largely ineffective under these conditions. The size of I(CRAC) to adenophostin A in weak Ca(2+) buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase ('SERCA') pumps with thapsi-gargin or enhancing mitochondrial Ca(2+) uptake, although blocking the mitochondrial Ca(2+) uniporter with Ruthenium Red did not suppress the activation of the current. Changing the levels of free ATP in the recording pipette did not enhance the size of I(CRAC) evoked by adenophostin A. We also examined two structurally distinct analogues of adenophostin A (manno-adenophostin and ribophostin), for which the affinities for the Ins(1,4,5)P(3) receptor are similar to that of Ins(1,4,5)P(3) in equilibrium binding experiments. Although these analogues were able to activate I(CRAC) to its maximal extent in strong buffer, ribophostin, but not manno-adenophostin, consistently activated the current in weak buffer. We conclude that adenophostin A and ribophostin are able to activate I(CRAC) in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P(3) and manno-adenophostin and is not related to equilibrium affinities. (+info)
The crystal structure and mechanism of 1-L-myo-inositol- 1-phosphate synthase.
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1-l-myo-Inositol-1-phosphate synthase catalyzes the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate (MIP), the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, intramolecular aldol cyclization, and reduction. We have determined the first crystal structure of MIP synthase. We present structures of both the NAD-bound enzyme and the enzyme bound to an inhibitor, 2-deoxy-glucitol-6-phosphate. While 58 amino acids are disordered in the unbound form of the enzyme in the vicinity of the active site, the inhibitor nucleates the folding of this domain in a striking example of induced fit, serving to completely encapsulate it within the enzyme. Three helices and a long beta-strand are formed in this process. We postulate a mechanism for the conversion based on the structure of the inhibitor-bound complex. (+info)
Two opines control conjugal transfer of an Agrobacterium plasmid by regulating expression of separate copies of the quorum-sensing activator gene traR.
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Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon. One copy, traR(noc), is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR(noc) abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR(acc), is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR(noc) with nox is unique, but the operon containing traR(acc) is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types. (+info)
Pentitol metabolism in Lactobacillus casei.
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Strains of Lactobacillus casei capable of growing on either ribitol or xylitol carry out a heterolactic fermentation producing ethanol, acetate, and a mixture of D- and L-lactate. Following conversion of the pentitols to ribulose 5-phosphate or xylulose 5-phosphate via enzymatic steps unique to these organisms, the intermediate products are further metabolized by enzymes of the pentose pathway. The initial enzymes of the pathway, i.e., pentitol:phosphoenolypyruvate phosphotransferase and penititol phosphate dehydrogenase, do not appear to be stringently regulated by glucose or intermediate products of glycolysis. (+info)
Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway.
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Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids. (+info)