A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.
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The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase. (+info)
Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli.
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Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP. (+info)
Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.
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Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type. (+info)
Photosystem II regulation of macromolecule synthesis in the blue-green alga Aphanocapsa 6714.
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Polymers synthesized by heterotrophically growing (glucose as carbon source) cultures of Aphanocapsa 6714 were compared with polymers synthesized in photosynthetically grown cultures. Loss of photosystem II by dark incubation, or inhibition of light-grown cells with the photosystem II-specific inhibitor dichlorophenylmethylurea, caused an 80 to 90% reduction in the rate of lipid and total ribonucleic acid synthesis, and more than a 90% reduction in the rate of protein synthesis. In contrast, glycogen synthesis was reduced only about 50% in dark cells and less than 30% in dichlorphenylmethylurea-inhibited cells. After longer heterotrophic growth, glycogen became the major component, whereas in photosynthetically grown cultures protein was the major constituent. 14C (from 14CO2 and/or [14C]glucose) assimilated into protein by heterotrophically grown cells was found in amino acids in nearly the same proportions as in photosynthetically grown cells. Thus, routes of biosynthesis available to autotropic cells were also available to heterotrophic cultures, but the supply of carbon precursors to those pathways was greatly reduced. The limited biosynthesis in heterotrophic cells was not due to a limitation for cellular energy. The adenylates were maintained at nearly the same concentrations (and hence the energy charge also) as in photosynthetic cells. The concentration of reduced nicotinamide adenine dinucleotide phosphate was higher in heterotrophic (dark) cells than in photosynthetic cells. From rates of CO2 fixation and/or glycogen biosynthesis it was determined that stationary-phase cells expended approximately 835, 165, and less than 42 nmol of adenosine 5'-triphosphate per mg (dry weight) of algae per 30 min during photosynthetic, photoheterotrophic, and chemoheterotrophic metabolism, respectively. Analysis of the soluble metabolite pools in dark heterotrophic cultures by double-labeling experiments revealed rapid equilibration of 14C through the monophosphate pools, but much slower movement of label into the diphosphate pools of fructose-1,6-diphosphate and sedoheptulose-1,7-diphosphate. Carbon did flow into 3-phosphoglycerate in the dark; however, the initial rate was low and the concentration of this metabolite soon fell to an undetectable level. In photosynthetic cells, 14C quickly equilibrated throughout all the intermediates of the reductive pentose cycle, in particular, into 3-phosphoglycerate. Analysis of glucose-6-phosphate dehydrogenase in cell extracts showed that the enzyme was very sensitive to product inhibition by reduced nicotinamide adenine dinucleotide. (+info)
Construction and characterization of Escherichia coli disruptants defective in the yaeM gene.
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Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli. (+info)
The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance.
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The TPS1 gene from Hansenula polymorpha, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of TPS1 rendered H. polymorpha cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells' ability to acquire thermotolerance. (+info)
In vivo gammadelta T cell priming to mycobacterial antigens by primary Mycobacterium tuberculosis infection and exposure to nonpeptidic ligands.
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BACKGROUND: The recognition of phosphorylated nonpeptidic microbial metabolites by Vgamma9Vdelta2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vgamma9Vdelta2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vgamma9Vdelta2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of gammadelta T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured. RESULTS: The Vgamma9Vdelta2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vgamma9Vdelta2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of gammadelta T cell activation in vivo. The in vivo reactivity of Vgamma9Vdelta2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vgamma9Vdelta2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children. CONCLUSIONS: The increased reactivity of Vgamma9Vdelta2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vgamma9Vdelta2 responses may be useful for monitoring the efficacy of antimycobacterial therapies. (+info)
Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol.
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2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphosphocytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-(14)C]2-C-methylerythritol was prepared from [2-(14)C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A gene from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants. (+info)