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At least 19 recordsLinked to original sources
PubMed1997

Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli.

Initiation of replication of the Escherichia coli chromosome is rendered temperature-sensitive by the dnaR130 mutation in the prs gene that encodes phosphoribosylpyrophosphate synthetase. The thermosensitivity of the dnaR mutant is suppressed by a spontaneous mutation in rpe, the gene encoding ribulosephosphate epimerase. Disruption of the rpe gene reverses the thermosensitive growth of the dnaR mutant. The rpe-disrupted dnaR mutant exhibits extensive DNA synthesis at low and high temperatures, as does the dnaR+ rpe disruptant and the dnaR+ rpe+ strain. The thermoresistant DNA synthesis in the rpe dnaR double mutant is dnaA dependent, because the dnaA167 mutation renders the synthesis thermosensitive. The rpe-knockout mutation abolishes the ability of the dnaA115 allele to complement the defect of the dnaA167 mutant with or without the dnaR mutation and diminishes the dnaR-complementing ability of the dnaR224 allele. These results show that the rpe product is involved in the functions of the products of both dnaA and dnaR for initiation of replication of the bacterial chromosome.

Bacterial Proteins↗
PubMed2000

Phosphoribulokinase: current perspectives on the structure/function basis for regulation and catalysis.

Phosphoribulokinase (PRK), an enzyme unique to the reductive pentose phosphate pathway of CO2 assimilation, exhibits distinctive contrasting properties when the proteins from eukaryotic and prokaryotic sources are compared. The eukaryotic PRKs are typically dimers of -39 kDa subunits while the prokaryotic PRKs are octamers of -32 kDa subunits. The enzymes from these two classes are regulated by different mechanisms. Thioredoxin of mediated thiol-disulfide exchange interconverts eukaryotic PRKs between reduced (active) and oxidized (inactive) forms. Allosteric effectors, including activator NADH and inhibitors AMP and phosphoenolpyruvate, regulate activity of prokaryotic PRK. The effector binding site has been identified in the high resolution structure recently elucidated for prokaryotic PRK and the7 apparatus for transmission of the allosteric stimulus has been identified. Additional contrasts between PRKs include marked differences in primary structure between eukaryotic and prokaryotic PRKs. Alignment of all available deduced PRK sequences indicates that less than 10% of the amino acid residues are invariant. In contrast to these differences, the mechanism for ribulose 1,5-biphosphate synthesis from ATP and ribulose 5-phosphate (Ru5P) appears to be the same for all PRKs. Consensus sequences associated with M++-ATP binding, identified in all PRK proteins, are closely juxtaposed to the residue proposed to function as general base catalyst. Sequence homology and mutagenesis approaches have suggested several residues that may potentially function in Ru5P binding. Not all of these proposed Ru5P binding residues are closely juxtaposed in the structure of unliganded PRK. Mechanistic approaches have been employed to investigate the amino acids which influence K(m Ru5P) and identify those amino acids most directly involved in Ru5P binding. PRK is one member of a family of phospho or sulfo transferase proteins which exhibit a nucleotide monophosphate kinase fold. Structure/function correlations elucidated for PRK suggest analogous assignments for other members of this family of proteins.

Catalytic Domain↗
PubMed1976

Bacterial yields on methanol, methylamine, formaldehyde, and formate.

Several bacteria utilizing C1-compounds as sole carbon sources were grown on these substrates in continuous culture. The molar yield values (g of cell dry wt/mol of substrate utilized) of bacteria which utilize C1-compounds via the ribulose monophosphate pathway were between 15.7 to 17.3 when grown on methanol; while the molar yield values of bacteria which use the serine pathway for the assimilation of C1-compounds varied between 9.8 and 13.1. The molar yield values of different bacteria which use the serine pathway decreased as the oxidation levels of the C1-growth substrates increased. On formaldehyde the values were between 7.2 to 9.6, whereas on formate the values varied from 3.3 to 6.9. It appears that bacteria utilize C1-compounds more efficiently via the ribulose monophosphate pathway than via the serine pathway. The oxidation step from methanol to formaldehyde (and from methylamine to formaldehyde) in the bacteria studied may be energy yielding. A comparison has been made between the experimental yield values obtained and theoretical values.

Formaldehyde↗
PubMed1978

Maintenance requirements for bacteria growing on C1-compounds.

The maintenance coefficient, ms (mmol substrate/g cell dry wt hr), of two distinct groups of C1-utilizing bacteria has been determined by growing the organisms in an aerobic continuous culture limited by different C1 growth substrates. For growth on methanol, ms = 2.5 +/- 0.3 for Pseudomonas C; 3.9 +/- 0.7 for Ps. methylotropha (these bacteria utilize methanol via the ribulose monophosphate pathway of formaldehyde fixation); 1.5 +/- 0.2 for Pseudomonas 1, and 2.3 +/- 0.4 for Pseudomonas 135 (the latter bacteria utilize C1-compounds via the serine pathway). For growth on formaldehyde, ms = 1.5 +/- 0.3 for Pseudomonas 1 and 2.7 +/- 0.7 for Pseudomonas 135, whereas on formate the values for ms are 1.0 +/- 0.2 and 4.4 +/- 1.3; respectively. Although the maintenance coefficients did not differ systematically between the two groups of bacteria, the maintenance requirements per generation of the serine pathway bacteria were considerably higher (8.7 vs. 3.9) owing to their slower growth rate. The maximum molar yield values, YMmax (g cell dry wt/mol substrate utilized), corrected for the maintenance energy of bacteria which utilize C1-compounds via the ribulose monophosphate pathway averaged 19.1 when grown on methanol, while the values for bacteria which use the serine pathway averaged 13.5. On formaldehyde an average value of 11.5 is obtained and on formate the average value was 7.4 in the serine pathway bacteria.

Adenosine Triphosphate↗
PubMed1980

A critical analysis of kinetic data of 3-hexulosephosphate synthases. Michaelis-Menten or complex characteristics.

Investigations of the 3-hexulosephosphate synthase (HPS) from different methylotrophic bacteria have revealed apparent discrepancies in kinetic behaviour. In all methanol-utilizing species investigated by us the kinetic characteristics showed intermediary plateau regions. Therefore, this behaviour is assumed to be a general feature of the HPS from all non-methane-utilizing methylotrophic bacteria. However, this assumption is in contrast to the results of other authors. Both for Methylomonas M15 (SAHM et al. 1976) and Methylomonas aminofaciens 77a (KATO et al. 1977, 1978) MICHAELIS-MENTEN kinetics of the HPS were stated. To check the validity of our assumption we have analyzed the kinetic data given by others. Indications of the existence of intermediary plateau regions could be found with the enzyme from Arthrobacter globiformis (BYKOVSKAYA and VORONKOV 1977) and Methylomonas aminofaciens 77a (KATO et al. 1978). Furthermore, biphasic ARRHENIUS plots indicate a multiple character of the HPS from these species as could already be demonstrated with the enzyme from Bacterium MB 58 and Pseudomonas oleovorans. In addition, causes which may obscure the detection of intermediary plateau regions are demonstrated.

Aldehyde-Lyases↗
PubMed1994

High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications.

Marine algae play an important role in removing carbon dioxide from the atmosphere. In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae. The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria. There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region. Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme. Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase. Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit. Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters. Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms. The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.

Amino Acid Sequence↗
PubMed1995

Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C].

UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.

Adenosine Triphosphate↗
PubMed1999

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.

Culture Media↗
PubMed2000

Proline alleviates salt-stress-induced enhancement in ribulose-1, 5-bisphosphate oxygenase activity.

Sodium chloride enhanced oxygenase activity while curtailing carboxylase activity of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) purified to electrophoretic homogeneity. Exposure to 200 mM NaCl brought about an increase in the potential of Rubisco to oxygenate RuBP by over 50%. On the other hand, proline suppressed both oxygenase as well as carboxylase activities of Rubisco. Interestingly, proline-induced suppression in oxygenase activity was significantly higher than that of carboxylase activity. Most amazingly, salt-stress-induced enhancement in oxygenase activity was fully alleviated by proline even when present at a concentration as low as 50 mM. The findings presented in this communication clearly demonstrate for the first time that stress-induced proline accumulation might have a critical role in lowering the loss in fixed carbon by curtailing salt-stress-promoted enhancement in oxygenase activity of Rubisco.

Fabaceae↗
PubMed1993

Crystallization and X-ray analysis of a multienzyme complex containing RUBISCO and RuBP.

Single crystals of a multienzyme complex isolated from spinach leaves, and containing RUBISCO bound to the substrate RuBP have been grown and characterized. The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 173 A, b = 134 A and c = 112 A, and contain two enzyme complex molecules in the unit cell. Diffraction data to 2.5 A resolution have been collected on the sychrotron source at the photon factory in Japan. Initial structure determination has been carried out using the molecular replacement method. The RUBISCO molecule in the complex has the normal L8S8 subunit configuration, and difference electron density is clearly observed for the other component enzymes and the RuBP substrate.

Crystallography, X-Ray↗
PubMed1997

The structure of the complex between rubisco and its natural substrate ribulose 1,5-bisphosphate.

The three-dimensional structure of the complex of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco; EC 4.1.1.39) from spinach with its natural substrate ribulose 1,5-bisphosphate (RuBP) has been determined both under activating and non-activating conditions by X-ray crystallography to a resolution of 2.1 A and 2.4 A, respectively. Under activating conditions, the use of calcium instead of magnesium as the activator metal ion enabled us to trap the substrate in a stable complex for crystallographic analysis. Comparison of the structure of the activated and the non-activated RuBP complexes shows a tighter binding for the substrate in the non-activated form of the enzyme, in line with previous solution studies. In the non-activated complex, the substrate triggers isolation of the active site by inducing movements of flexible loop regions of the catalytic subunits. In contrast, in the activated complex the active site remains partly open, probably awaiting the binding of the gaseous substrate. By inspection of the structures and by comparison with other complexes of the enzyme we were able to identify a network of hydrogen bonds that stabilise a closed active site structure during crucial steps in the reaction. The present structure underlines the central role of the carbamylated lysine 201 in both activation and catalysis, and completes available structural information for our proposal on the mechanism of the enzyme.

Calcium↗
PubMed1978

Regulation of photosynthetic carbon assimilation.

It may be concluded that the conversion of PGA to DPGA plays a key role in induction and in the regulation of cycle activity. The high concentrations of PGA in actively photosynthesizing chloroplasts reflect this role and the control exerted by adenylate ratios. Thus the cycle can operate at its maximum rate only in the presence of high PGA and low ribulose 5-phosphate concentrations. Once induction is complete, the reductive pentose phosphate pathway will continue to function at its maximum rate if sink activity within the cytoplasm makes available sufficient Pi to support rapid export of triose phosphate. If triose phosphate tends to build up in the straoma, it will favor pentose monophosphate accumulation. A relative excess of ribulose 5-phosphate would, in turn, inhibit PGA reduction (and hence its own formation) by drawing too heavily on the available ATP.

Adenosine Diphosphate↗
PubMed1990

D-ribulokinase from Klebsiella pneumoniae for continuous production of D-(-)-ribulose-5-phosphate.

The production of D-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to D-ribulose was catalysed by D-ribulokinase isolated from Klebsiella pneumoniae. For production of D-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for D-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of D-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of D-ribulose-5-phosphate l -1 per day were reached.

Adenosine Triphosphate↗
PubMed1995

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants.

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplast-located GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T1 seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T1 progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose-1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO2 assimilation and other gas-exchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO2 assimilation was not affected until RuBP levels dropped to 30-40% of WT levels. That is, the CO2 assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol.(mol site)-1 while the ratio for WT plants was 4-5 mol.m(mol site)-1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO2 partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO2 and showed a normal decline with increases in CO2 partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-exchange and chlorophyll alpha fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO2 partial pressure in the antisense plants than in WT plants.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1978

Glucose-6-phosphate dehydrogenase of Anabaena sp. Kinetic and molecular properties.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three pincipal forms, with approximate molecular weights of 120 000 (M1), 240 000 (M2) and 345 000 (M3). The relative catalytic activities are: M1 less than M2 less than M3. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M1 form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP+), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M1 form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M1 form is favored at pH values above 7; a pH of 6.5 is optimal for maintenance of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5'-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.

Adenosine Triphosphate↗