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PubMed2000

Analysis of two formaldehyde oxidation pathways in Methylobacillus flagellatus KT, a ribulose monophosphate cycle methylotroph.

The roles of cyclic formaldehyde oxidation via 6-phosphogluconate dehydrogenase and linear oxidation via the tetrahydromethanopterin (H4MPT)-linked pathway were assessed in an obligate methylotroph, Methylobacillus flagellatus KT, by cloning, sequencing and mutating two chromosomal regions containing genes encoding enzymes specifically involved in these pathways: 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase and methenyl H4MPT cyclohydrolase (gndA, zwf and mch). No null mutants were obtained in gndA or zwf, implying that the cyclic oxidation of formaldehyde is required for C1 metabolism in this obligate methylotroph, probably as the main energy-generating pathway. In contrast, null mutants were generated in mch, indicating that the H4MPT-linked pathway is dispensable. These mutants showed enhanced sensitivity to formaldehyde, suggesting that this pathway plays a secondary physiological role in this methylotroph. This function is in contrast to Methylobacterium extorquens AM1, in which the H4MPT-linked pathway is essential.

Cloning, Molecular↗
PubMed2002

Sensitivity of photosynthesis in a C4 plant, maize, to heat stress.

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38 degrees C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30 degrees C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO(2) assimilation, the maximum quantum yield of photosystem II (F(v)/F(m)) was relatively insensitive to leaf temperatures up to 45 degrees C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40 degrees C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45 degrees C. The activation state of Rubisco decreased at temperatures exceeding 32.5 degrees C, with nearly complete inactivation at 45 degrees C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30 degrees C.

Acclimatization↗
PubMed2002

CO(2)-triggered chloride release from guard cells in intact fava bean leaves. Kinetics of the onset of stomatal closure.

The influence of CO(2) on Cl(-) release from guard cells was investigated within the intact leaf by monitoring the Cl(-) activity in the apoplastic fluid of guard cells with a Cl(-)-sensitive microelectrode. In illuminated leaves adapted to a CO(2) concentration within the cuvette of 350 microL L(-1), an increase of 250 microL L(-1) CO(2) triggered a transient rise in the apoplastic Cl(-) activity from 3 to 14 mM within 10 min. This Cl(-) response was similar to the Cl(-) efflux evoked by turning off the light, when the substomatal CO(2) was kept constant (CO(2) clamp). Without CO(2) clamp, substomatal CO(2) increased by 120 microL L(-1) upon "light off." The response to an increase in CO(2) within the cuvette from 250 to 500 microL L(-1) in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 microL L(-1) in the light. No Cl(-) efflux was triggered by 2-min CO(2) pulses (150-800 microL L(-1)). After a switch from 350 microL L(-1) to CO(2)-free cuvette air, the guard cells were less sensitive to a rise in CO(2) and to light off, but the sensitivity to both stimuli partially recovered. Changes in CO(2) also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl(-) responses, and which also promptly occurred when CO(2) did not initiate Cl(-) efflux. The comparatively slow activation of Cl(-) efflux by CO(2) indicates that an intermediate effector derived from CO(2) has to accumulate to fully activate plasma membrane anion channels of guard cells.

Adaptation, Physiological↗
PubMed2002

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants.

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.

Aldose-Ketose Isomerases↗
PubMed2005

The regulation of Rubisco activity in response to variation in temperature and atmospheric CO2 partial pressure in sweet potato.

The temperature response of net CO(2) assimilation rate (A), the rate of whole-chain electron transport, the activity and activation state of Rubisco, and the pool sizes of ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglyceric acid (PGA) were assessed in sweet potato (Ipomoea batatas) grown under greenhouse conditions. Above the thermal optimum of photosynthesis, the activation state of Rubisco declined with increasing temperature. Doubling CO(2) above 370 mubar further reduced the activation state, while reducing CO(2) by one-half increased it. At cool temperature (<16 degrees C), the activation state of Rubisco declined at CO(2) levels where photosynthesis was unaffected by a 90% reduction in O(2) content. Reduction of the partial pressure of CO(2) at cool temperature also enhanced the activation state of Rubisco. The rate of electron transport showed a pronounced temperature response with the same temperature optimum as A at elevated CO(2). RuBP pool size and the RuBP-to-PGA ratio declined with increasing temperature. Increasing CO(2) also reduced the RuBP pool size. These results are consistent with the hypothesis that the reduction in the activation state of Rubisco at high and low temperature is a regulated response to a limitation in one of the processes contributing to the rate of RuBP regeneration. To further evaluate this possibility, we used measured estimates of Rubisco capacity, electron transport capacity, and the inorganic phosphate regeneration capacity to model the response of A to temperature. At elevated CO(2), the activation state of Rubisco declined at high temperatures where electron transport capacity was predicted to be limiting, and at cooler temperatures where the inorganic phosphate regeneration capacity was limiting. At low CO(2), where Rubisco capacity was predicted to limit photosynthesis, full activation of Rubisco was observed at all measurement temperatures.

Carbon Dioxide↗
PubMed2001

A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism.

Transketolase (TK) catalyzes reactions in the Calvin cycle and the oxidative pentose phosphate pathway (OPPP) and produces erythrose-4-phosphate, which is a precursor for the shikimate pathway leading to phenylpropanoid metabolism. To investigate the consequences of decreased TK expression for primary and secondary metabolism, we transformed tobacco with a construct containing an antisense TK sequence. The results were as follows: (1) a 20 to 40% reduction of TK activity inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis. The inhibition of photosynthesis became greater as irradiance increased across the range experienced in growth conditions (170 to 700 micromol m(-2) sec(-1)). TK almost completely limited the maximum rate of photosynthesis in saturating light and saturating CO(2). (2) Decreased expression of TK led to a preferential decrease of sugars, whereas starch remained high until photosynthesis was strongly inhibited. One of the substrates of TK (fructose-6-phosphate) is the starting point for starch synthesis, and one of the products (erythrose-4-phosphate) inhibits phosphoglucose isomerase, which catalyzes the first reaction leading to starch. (3) A 20 to 50% decrease of TK activity led to decreased levels of aromatic amino acids and decreased levels of the intermediates (caffeic acid and hydroxycinnamic acids) and products (chlorogenic acid, tocopherol, and lignin) of phenylpropanoid metabolism. (4) There was local loss of chlorophyll and carotene on the midrib when TK activity was inhibited by >50%, spreading onto minor veins and lamina in severely affected transformants. (5) OPPP activity was not strongly inhibited by decreased TK activity. These results identify TK activity as an important determinant of photosynthetic and phenylpropanoid metabolism and show that the provision of precursors by primary metabolism colimits flux into the shikimate pathway and phenylpropanoid metabolism.

Antisense Elements (Genetics)↗
PubMed2004

Metal sites in 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate.

The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of ribulose 5-phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A.

Binding Sites↗
PubMed2005

Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co-inducer recognition.

CbbR is a LysR-type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO2 fixation, in Rhodobacter sphaeroides. LTTR proteins often require a co-inducer to regulate transcription. Previous studies suggested that ribulose 1,5-bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/cbb(I) promoter complex. To define and analyse the co-inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three-dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co-inducer binding.

Amino Acid Substitution↗
PubMed2006

Can improvement in photosynthesis increase crop yields?

The yield potential (Yp) of a grain crop is the seed mass per unit ground area obtained under optimum growing conditions without weeds, pests and diseases. It is determined by the product of the available light energy and by the genetically determined properties: efficiency of light capture (epsilon i), the efficiency of conversion of the intercepted light into biomass (epsilon c) and the proportion of biomass partitioned into grain (eta). Plant breeding brings eta7 and epsilon i close to their theoretical maxima, leaving epsilon c, primarily determined by photosynthesis, as the only remaining major prospect for improving Yp. Leaf photosynthetic rate, however, is poorly correlated with yield when different genotypes of a crop species are compared. This led to the viewpoint that improvement of leaf photosynthesis has little value for improving Yp. By contrast, the many recent experiments that compare the growth of a genotype in current and future projected elevated [CO2] environments show that increase in leaf photosynthesis is closely associated with similar increases in yield. Are there opportunities to achieve similar increases by genetic manipulation? Six potential routes of increasing epsilon c by improving photosynthetic efficiency were explored, ranging from altered canopy architecture to improved regeneration of the acceptor molecule for CO2. Collectively, these changes could improve epsilon c and, therefore, Y p by c. 50%. Because some changes could be achieved by transgenic technology, the time of the development of commercial cultivars could be considerably less than by conventional breeding and potentially, within 10-15 years.

Biomass↗
PubMed2006

Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures.

Recently, several studies reported that the optimum temperature for the initial slope [IS(Ci)] of the light-saturated photosynthetic rate (A) versus intercellular CO2 concentration (Ci) curve changed, depending on the growth temperature. However, few studies compare IS(Ci) with ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) properties. Here, we assessed Rubisco activation state and in vitro Rubisco kinetics, the main determinants of IS(Ci), in spinach leaves grown at 30/25 [high temperature (HT)] and 15/10 degrees C [low temperature (LT)]. We measured Rubisco activation state and A at a CO2 concentration of 360 microL L(-1) (A360) at various temperatures. In both HT and LT leaves, the Rubisco activation state decreased with increasing temperatures above the optimum temperatures for A360, while the activation state remained high at lower temperatures. To compare Rubisco characteristics, temperature dependences of the maximum rate of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax), specificity factor (Sc/o) and thermal stability were examined. We also examined Vcmax, and thermal stability in the leaves that were transferred from HT to LT conditions and were subsequently kept under LT conditions for 2 weeks (HL). Rubisco purified from HT, LT and HL leaves are called HT, LT and HL Rubisco, respectively. Thermal stabilities of LT and HL Rubisco were similar and lower than that of HT Rubisco. Both Vcmax and Sc/o in LT Rubisco were higher than those of HT Rubisco at low temperatures, while these were lower at high temperatures. Vcmax in HL Rubisco were similar to those of LT Rubisco at low temperatures, and to those of HT Rubisco at high temperatures. The predicted photosynthetic rates, taking account of the Rubisco kinetics and the Rubisco activation state, agreed well with A360 in both HT and LT leaves. This study suggests that photosynthetic performance is largely determined by the Rubisco kinetics at low temperature and by Rubisco Kinetics and the Rubisco activation state at high temperature.

Enzyme Activation↗
PubMed1997

Memory and imprinting effects in multienzyme complexes--II. Kinetics of the bienzyme complex from Chlamydomonas reinhardtii and hysteretic activation of chloroplast oxidized phosphoribulokinase.

Oxidized, free, stable phosphoribulokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s(-1)/site). However, when it was bound to glyceraldehyde-3-phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s(-1)/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s(-1)/site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and k(cat)) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5-phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5-phosphate tended to stabilize this complex. Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde-3-phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde-3-phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde-3-phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity. The potential biological significance of these effects remains to be discovered.

Adenosine Triphosphate↗
PubMed1980

Biochemical genetics of the pentose phosphate cycle: human ribose 5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE).

1. Staining procedures are described for the detection after starch-gel electrophoresis of ribose-5-phosphate isomerase (RPI) and ribulose 5-phosphate 3-epimerase (RPE). 2. Both RPI and RPE were detected in all human tissues including red cells, lymphocytes and fibroblasts. 3. No evidence was found for more than one structural gene locus for either enzyme. 4. No allelic variants of either enzyme were found in erythrocyte lysates from over 200 unrelated individuals. 5. Preliminary data are presented which suggest that differences in tissue RPE isozyme patterns may be due to endogenous proteolytic activity. 6. Electrophoretic analysis of RPE and RPI isozyme patterns in extracts of man/mouse hybrid cells indicates that RPE is probably a dimer and RPI may also be polymeric.

Animals↗
PubMed1986

Noninvasive in vivo demonstration of 2-fluoro-2-deoxy-D-glucose metabolism beyond the hexokinase reaction in rat brain by 19F nuclear magnetic resonance spectroscopy.

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.

Animals↗
PubMed1999

Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a.

The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6-phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects.

Aldehyde-Lyases↗
PubMed1990

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.

Bacteria↗
PubMed2006

The ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis.

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

Aldehyde-Lyases↗