The genome of 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 contains two genes for malic enzymes. One uses NAD⁺ as a cofactor (𝘥𝘮𝘦) and one utilizes NADP⁺ (𝘵𝘮𝘦). The two enzymes have been purified and the genes cloned and sequenced. Loss of TME enzyme function gives no detectable phenotype in either 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture or in bacteroids. Loss of DME function gives no detectable phenotype in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture but does result in bacteroids that are unable to fix nitrogen (Fix⁻). Expression of 𝘵𝘮𝘦 is reduced in bacteroids whereas 𝘥𝘮𝘦 expression remains unchanged. In order to overexpress 𝘵𝘮𝘦 in bacteroids a fusion gene was constructed with the 𝘥𝘮𝘦 promoter driving expression of the 𝘵𝘮𝘦 structural gene (𝘥𝘵𝘮𝘦). The 𝘥𝘵𝘮𝘦 gene was expressed and functional in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, but alfalfa plants inoculated with strains expressing only the 𝘥𝘵𝘮𝘦 gene were Fix⁻. In addition the NAD⁺-dependent malic enzyme gene from 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘣𝘰𝘷𝘪𝘴 (𝘮𝘢𝘦𝘌) was similarly cloned downstream of the 𝘥𝘮𝘦 promoter. The fusion gene 𝘥𝘮𝘢𝘦𝘌 was expressed in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, surprisingly plants inoculated with strains expressing only the 𝘥𝘮𝘢𝘦𝘌 gene showed a Fix⁻ phenotype. A truncated 𝘥𝘮𝘦 gene was constructed which contained only the N-terminal, malic enzyme domain of the protein (𝘥𝘮𝘦Δ𝘗𝘴𝘵). The truncated enzyme was expressed and active in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture and gave a Fix⁺ phenotype when inoculated onto alfalfa plants. / Thesis / Master of Science (MS)

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

Thesis (Ph.D.)--University of Wollongong, 2008. / Typescript. Includes bibliographical references.

Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

The diploma thesis is focused on the study of the influence of cultivation conditions and arrangement for the production of extracellular polymeric substances by using yeast-like microorganism Aureobasidium pullulans. In the theoretical part a brief description of A. pullulans, its use in biotechnology and produced exobiopolymers, especially pullulan and poly-L-malic acid are presented. The first aim of the experimental part was to set the most appropriate cultivation conditions for A. pullulans CCM 8182. Growth and production properties in optimum conditions were compared with cultivation on waste substrates - oat bran, buckwheat husks, apple fiber and others. Waste substrates can be used as cheap nutrient sources which enable reducing cost of potential biotechnological production. As a further part of this work, optimization of HPLC/RI method for analysis of exobiopolymers has been done. Optimal mobile phase composition and chromatography conditions were proposed. Column Roa organic acid H+ was the most suitable for simultaneous separartion of glucose and malic acid. Before HPLC analysis hydrolysis of polymers was done. Sulphuric acid (5 mmol/L) was used as a mobile phase at flow rate 0.5 mL/min and temperature 60 °C. The highest production of pullulan occurred using oat bran as a substarate (13.03 g/L) at an initial pH 7.5. Maximum production of poly-L-malic acid was observed during the cultivation on apple peels (2.89 g/L) at pH 6. It was found that the higher production of poly-L-malic acid occurred at pH 6, while higher production of pullulan was at pH 7.5.

Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a secondary 13-C effect in the hydride transfer step. This phenomenon is also applicable to the modified enzyme with NAD as the substrate.

Thirty-two lactating cows were assigned at random to four treatments of malic acid to determine if these levels had an effect on milk production, milk composition, feed intake, and efficiency of feed utilization. Malic acid allotment for each treatment consisted of 1) 15.4, 2) 11.6, 3) 7.7, and 4) 0 grams of malic acid fed per kilogram of concentrate. Concentrate was fed according to production at a rate of one kilogram per two kilograms of milk in excess of 9.1 kilograms of milk per cow daily. Alfalfa hay was fed free choice and corn silage at a rate of 11.4 kilograms daily. The cows were on the trial for 8 weeks. Intake of concentrates, silage, dry matter, and digestible energy was highest for cows receiving the 11.6 g level of malic acid. These intakes were significantly higher than for the 7.7 g level but not for the other treatments. However, cows on the 7.7 g level consumed only slightly less feed than control cows. There was no significant effect on hay or crude protein intake. Production of total milk, fat corrected milk, and milk fat was significantly higher for cows receiving the 11.6 g level of malic acid than from the 7.7 g level or control cows. Production of protein solids-not-fat was significantly higher for the 11.6 g level than from the 7.7 g level and approached this level of significance when the 11.6 g level was compared to the controls. Cows receiving the 11.6 g level of malic acid were significantly more efficient in converting dry matter or digestible energy from feed into milk than were the controls. Intakes and production of cows on the 15.4 g level was slightly less than for the 11.6 g level.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13458号 / 論農博第2899号 / 新制||農||1088(附属図書館) / 学位論文||R3||N5362(農学部図書室) / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p> Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate with the simultaneous reduction of a nicotinamide cofactor. It was previously reported that the nitrogen-fixing bacterium, Sinorhizobium meliloti, has two malic enzymes, a diphosphopyridine-dependent malic enzyme (DME) and a triphosphopyridine-dependent malic enzyme (TME). The dme gene is essential for symbiotic nitrogen-fixation in alfalfa root nodules and this symbiotic requirement cannot be met through increased expression of tme. In order to determine if a metabolic difference exists between the dme and tme mutants which might explain the symbiotic phenotypes, we conducted an analysis of intracellular and extracellular polar metabolomes. Differences noted between the intracellular profiles of the dme and tme mutant strains hinted at osmotic stress or a disturbance in central carbon metabolism. Extracellular studies indicated that dme mutant cells excreted at least 10-fold greater concentrations of both malate and fumarate. When considered together, the metabolic data implies that the DME enzyme is primarily responsible for the conversion of malate to pyruvate to generate acetyl-CoA whereas the TME enzyme must serve a secondary function within the cell.</p> <p> While the C-terminal 320 amino acid regions from both DME and TME are similar in sequence to phosphotransacetylase enzymes, enzyme assays with DME and TME proteins have failed to detect PTA activity. Here we report that the chimeric malic enzyme structure is conserved among various gram negative bacteria including Agrobacterium tumefaciens, Escherichia coli, Bradyrhizobium japonicum and Porphyromonas gingivalis. Moreover these chimeric proteins are also present in the archaebacteria. Halobacterium salinarum and Haloarcula marismortui. To further our understanding of the functions of DME and TME in S. meliloti, we have fused protein domains from DME to an affinity tag consisting of strepII and a calmodulin binding peptide. To identify proteins interacting with this fusion, we expressed these protein fusion constructs in S. meliloti, prepared extracts containing the soluble proteins and passed these through tandem affinity chromatography columns. All proteins that coeluted with the fusion proteins appeared to be interacting with antibodies specific for the DME protein and so may have been aggregates or break-down products of DME.</p> / Thesis / Master of Science (MSc)