The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

The synergistic decarboxylation of glyoxylate and 2-oxoglutarate by an enzyme system from pig-liver mitochondria

1. An enzyme system that catalyses a synergistic decarboxylation of glyoxylate and 2-oxoglutarate has been purified from pig-liver mitochondria. 2. The purified system is specific for glyoxylate and 2-oxoglutarate as substrates, although in earlier stages of purification glycine and l-glutamate are also active. 3. The reaction is inhibited strongly by EDTA and N-ethylmaleimide. Substrate analogues, present at concentrations equimolar with respect to the substrates, are not effective as inhibitors. 4. The reaction proceeds in the absence of added cofactors. Magnesium chloride, mercaptoethanol and sucrose stimulate the reaction, and stabilize the activity of the enzyme. 5. The pH optimum of the reaction is 7·0. The K_m values of glyoxylate and 2-oxoglutarate, at saturating concentration of the corresponding co-substrate, are 16mm and 3·6mm respectively. 6. Isotopic work with specifically labelled [¹⁴C]glyoxylate and 2-oxo[¹⁴C]-glutarate suggests that the enzyme system catalyses an initial condensation of glyoxylate and 2-oxoglutarate that results in, or leads to, release of C-1 of both substrates as carbon dioxide. C-2 of glyoxylate and C-5 of 2-oxoglutarate do not appear as carbon dioxide. 7. The stoicheiometry of the reaction is complex. During the initial stages of the reaction, more carbon dioxide is recovered from 2-oxoglutarate than from glyoxylate. Subsequently, there is a disproportionate increase with time of carbon dioxide evolution from the carboxyl group of glyoxylate. The excess of decarboxylation of glyoxylate over 2-oxogluturate is further increased by treatment of reaction products with acid.     

The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration

Large quantities of CO₂ are released within many photosynthesizing tissues in the light by the process of photorespiration. This CO₂ arises largely from the carboxylcarbon atom of glycolate, which is synthesized in chloroplasts during photosynthesis. Glyoxylate is then produced by the glycolate oxidase reaction. The glyoxylate may be directly decarboxylated to CO₂, but some investigators believe the glyoxylate must first be converted to glycine before CO₂ is released during photorespiration. Spinach chloroplasts with their envelope membranes removed in dilute buffer solution have now been shown to carry out the oxidative decarboxylation of [1-¹⁴C]glyoxylate, in the presence of light and manganous ions in an atmosphere containing oxygen, to yield 1 mole each of ¹⁴CO₂ and formate. Rates of enzymatic decarboxylation exceeding 50 μmoles of ¹⁴CO₂ mg chlorophyll⁻¹ hr⁻¹ were obtained at pH 7.6; hydrogen peroxide is probably the oxidant in the reaction. Heated chloroplasts are inactive under the standard conditions and there is an almost absolute requirement for each of the components listed above. Conditions for some other nonenzymatic decarboxylations of glyoxylate have also been described. [1-¹⁴C]Glycine is decarboxylated by the enzymatic system at only 1% of the rate of [1-¹⁴C]glyoxylate. Maize chloroplast preparations are much less active than spinach chloroplasts. The high rates of CO₂ produced by the spinach system directly from glyoxylate, as well as the need for light and oxygen, suggest that this reaction functions in photorespiration, and that CO₂ arises during photorespiration without glycine as a mandatory intermediate.     

The importance of glyoxylate in amino acid biosynthesis in plants

1. [¹⁴C₂]Glyoxylate was rapidly metabolized by carrot storage tissues, pea leaves, pea cotyledons, sunflower cotyledons, corn coleoptiles, corn roots and pea roots. In many tissues over 70% of the supplied [¹⁴C₂]glyoxylate was utilized during the 6hr. experimental periods. 2. In all tissues, the chief products of [¹⁴C₂]-glyoxylate metabolism were carbon dioxide, glycine and serine. In several of the tissues, there was also a considerable incorporation of the label into the organic acids, particularly into glycollate. 3. Degradations of the labelled serine produced during [¹⁴C₂]glyoxylate metabolism showed that glyoxylate carbon was incorporated into all three positions of the serine molecule. 4. The results are interpreted as indicating that glyoxylate is utilized by the tissues by pathways involving transamination, transmethylation, reduction and oxidative decarboxylation of the supplied glyoxylate.     

Oxidation of one-carbon compounds to formate and carbon dioxide in rat liver mitochondria.

In rat liver mitochondria, swollen with phosphate and supplemented with NAD⁺, the oxidation of the methyl carbon of sarcosine to formate is enhanced by the addition of NADP⁺. No carbon dioxide is formed. Formaldehyde and serine, which are the only oxidation products of the methyl group in the absence of the pyridine nucleotides, are decreased by an amount equal to the formate produced. Carbon dioxide, as well as formate, is produced when the mitochondria are treated with EDTA, even without the addition of the pyridine nucleotides. When the mitochondria are exposed to pyrophosphate without added NAD⁺ and/or NADP⁺, all of the oxidized sarcosine-methyl can be recovered as formate, [3-C]serine, and carbon dioxide. Formaldehyde accumulates only if the system is supplemented with Mg²⁺. In the presence of NADP⁺ or the combined pyridine nucleotides, serine accumulation is depressed by an amount equal to the increase in carbon dioxide production. Both carbons of glycine and the 3-C of serine can also be oxidized to carbon dioxide in the pyrophosphate-treated mitochondria. The oxidation of the methyl carbon of S-adenosylmethionine to formaldehyde, [3-C]serine, formate, and carbon dioxide requires a whole homogenate supplemented with glycine. Neither exogenous formaldehyde nor formate is oxidized to carbon dioxide in any of the mitochondrial systems capable of converting sarcosine-methyl to carbon dioxide. Under conditions in which [N⁵,N¹⁰-¹⁴C-methylene]- and [N¹⁰-¹⁴C-formyl]tetrahydrofolate can be isolated as intermediate products of [¹⁴CH₃]sarcosine, exogenous [N⁵,N¹⁰-¹⁴C-methylene]tetrahydrofolate can also be converted to [3-¹⁴C]serine, [¹⁴C]formate, and [¹⁴C]carbon dioxide.     

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD⁺, about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO₂ was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H₂O₂, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25–30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO₂ from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.     

Glyoxylate decarboxylation during glycollate oxidation by pea leaf extracts: significance of glyoxylate and extract concentrations

Hydrogen peroxide-dependent glyoxylate decarboxylation occurring during glycollate oxidation by pea leaf extracts (Pisum sativum L.) has been studied in relation to the effects of glyoxylate and extract concentration. With a saturating concentration of glycollate, decarboxylation was greatly stimulated by raising the glyoxylate concentration; at 30°C and with approx. 0.04 nkat of glycollate oxidase (as leaf extract) in the reaction mixture, CO₂ release in the presence of 5 mM glycollate and 5 mM glyoxylate was equal to about 45% of glycollate oxidation. However, CO₂ release at these substrate concentrations was not linearly proportional to the amount of extract supplied and was equal to a diminishing proportion of glycollate oxidation as the amount of extract was increased. This was shown to be due to the low affinity of catalase for H₂O₂, so that the endogenous catalase was able to destroy a larger proportion of the H₂O₂ generated at higher extract concentrations. It is argued that although at high glycoxylate concentrations (5–10 mM) in vitro, glyoxylate decarboxylation can be made to equal more than a third of the glycollate oxidised, less than 10% of the glyoxylate generated in vivo is likely to be decarboxylated in peroxisomes where high concentrations of glycollate oxidase and catalase are localised and where high concentrations of glyoxylate are unlikely to be maintained.Abbreviation PHMS pyrid-2-yl--hydroxymethane sulphonic acid     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Influence of carbon availability on the production of NO, N2O, N2 and CO2 by soil cores during anaerobic incubation

Net productions of permanent soil atmosphere gases (N₂, CO₂, O₂) and temporary gases (N₂O, NO) were monitored in soil cores using a non-interfering, fully automated measuring technique allowing highly time resolved measurements over prolonged periods. The influence of changes in available organic carbon on CO₂, N₂O, NO and N₂ production was studied by changing the soil carbon content through aerobic preincubations of different length, up to 21 days.The aerobic preincubation caused an increase in NO₃ ^- concentration and a decrease in available carbon content. Available carbon content dominated both CO₂ and total N gas (N₂+N₂O+NO) production during anaerobiosis. Both CO₂ and total N gas production rates decreased with increasing length of the previous aerobic preincubation, this in spite of the higher initial NO₃ ^- concentration.Total denitrification rates were closely related to the anaerobic CO₂ production rates. No relation was found between water soluble carbon content and total denitrification. The N₂O/N₂ ratio could be explained by an interaction of carbon availability, NO₃ ^- concentration and enzyme status. Net N₂O consumption was monitored. The balance between cumulative total N gas production and NO₃ ^- consumption varied according to the different treatments. Cumulative N₂O production exceeded cumulative N₂ production for 0 up to 5 days.     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

beta-Decarboxylation of L-aspartic acid: a metal chelate-catalyzed reaction.

beta-decarboxylation of L-aspartic acid was observed in the system, pyridoxal: L-aspartic acid:aluminum(III), 1:100:1 when heated at 80 degrees for three hours. This reaction was followed by electronic spectroscopy and showed quantitative conversion of pyridoxal to pyridoxamine indicating decarboxylation of the ketimine. alpha-Methyl-L-aspartic acid was not decarboxylated indicating the presence of the alpha-proton and prior transamination as requirements for decarboxylation. When pyridoxamine and oxalo-2-propionic acid were reacted at pD 4.60, product analysis by nmr showed the presence of pyridoxamine and alpha-ketobutyric acid, indicating hydrolysis of the decarboxylated ketimine. Decarboxylation was fast compared to spontaneous decarboxylation. A mechanism is proposed for non-enzymatic decarboxylation and the previously suggested mechanism for the inactivation of the enzyme aspartate beta-decarboxylase is discussed.     

Unexpected isomeric equilibrium in pyridoxamine Schiff bases

Pyridoxamine is a vitamin B₆ derivative involved in biological reactions such as transamination, and can also act as inhibitor in protein glycation. In both cases, it has been reported that Schiff base formation between pyridoxamine and carbonyl compounds is the main step. Nevertheless, few studies on the Schiff base formation have been reported to date. In this work, we conduct a comparative study of the reaction of pyridoxamine and 4-picolylamin (a pyridoxamine analog) with various carbonyl compounds including propanal, formaldehyde and pyruvic acid. Based on the results, 4-picolylamin forms a Schiff base as end-product of its reactions with propanal and pyruvic acid, but a carbinolamine with formaldehyde. On the other hand, pyridoxamine forms a Schiff base with the three reagents, but the end-product is in equilibrium with its hemiaminal form, which results from the attack of the phenolate ion of the pyridine ring on the imine carbon. This isomeric equilibrium should be considered in studying reactions involving amine derivatives of vitamin B₆.     

Extremely high activities of phosphoenolpyruvate carboxylase in thermogenic tissues of Araceae

Phosphoenolpyruvate carboxylase activity in extracts of a wide range of thermogenic tissues of the Araceae was shown to be in the range 10–100 mol g^-1 fresh weight min^-1 (0.5–3.7 mol mg^-1 protein min^-1). Such high activities were not found in non-thermogenic tissues of the Araceae or in thermogenic tissues of Aristolochia brasiliensis Mart. and Zucc., Victoria amazonica Schomb. and Encephalartos barteri Carruth. During development and thermogenesis in the club of Arum maculatum L. the high activities of the carboxylase did not lead to any marked accumulation of citrate, isocitrate, 2-oxoglutarate, fumarate, malate and oxaloacetate. Clubs of Arum maculatum and of Arum italicum Miller readily fixed ¹⁴CO₂ in the dark, mostly into aspartate, malate, alanine and glutamate. Pulse and chase experiments showed that most of the fixed carbon was very rapidly metabolized to CO₂. The detailed distribution suggest that this occurred largely by decarboxylation of C-4 acids. It is suggested that thermogenic tissues of the Araceae are characterized by very high activities of phosphoenolpyruvate carboxylase, and that in vivo this leads to synthesis of C-4 acids which are promptly decarboxylated.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

温带针阔混交林土壤碳氮气体通量的主控因子与耦合关系

中高纬度森林地区由于气候条件变化剧烈,土壤温室气体排放量的估算存在很大的不确定性,并且不同碳氮气体通量的主控因子与耦合关系尚不明确。以长白山温带针阔混交林为研究对象,采用静态箱-气相色谱法连续4a(2005—2009年)测定土壤二氧化碳(CO2)、甲烷(CH4)和氧化亚氮(N2O)净交换通量以及温度、水分等相关环境因子。研究结果表明:温带针阔混交林土壤整体上表现为CO2和N2O的排放源和CH4的吸收汇。土壤CH4、CO2和N2O通量的年均值分别为-1.3 kg CH4hm-2a-1、15102.2 kg CO2hm-2a-1和6.13 kg N2O hm-2a-1。土壤CO2通量呈现明显的季节性规律,主要受土壤温度的影响,水分次之;土壤CH4通量的季节变化不明显,与土壤水分显著正相关;土壤N2O通量季节变化与土壤CO2通量相似,与土壤水分、温度显著正相关。土壤CO2通量和CH4通量不存在任何类型的耦合关系,与N2O通量也不存在耦合关系;土壤CH4和N2O通量之间表现为消长型耦合关系。这项研究显示温带针阔混交林土壤碳氮气体通量主要受环境因子驱动,不同气体通量产生与消耗之间存在复杂的耦合关系,下一步研究需要深入探讨环境变化对其耦合关系的影响以及内在的生物驱动机制。     

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release

Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO₂ was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.     

Chemical,genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome

Pyridoxal‐5′‐phosphate (vitamin B₆) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B₆de novo and rely on uptake of B₆ vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg⁻¹ and a K_m^app with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.     

Contribution of plants to N 2 O emissions in soil-winter wheat ecosystem: pot and field experiments

Outdoor pot and field experiments were conducted to assess the role of growing plants in agricultural ecosystem N₂O emissions. N₂O emissions from plants were quantified as the difference in soil-crop system N₂O emissions before and immediately after cutting plants during the main growth stages in 2001–02 and 2002–03 winter wheat seasons. Emissions of N₂O from plants depended on biomass within the same plant developmental status. Field results indicated that the seasonal contribution of N₂O emissions from plants to ecosystem fluxes averaged 25%, ranging from 10% at wheat tillering to 62% at the heading stage. The fluxes of N₂O emissions from plants varied between 0.3 and 3.9 mg N₂O-N m⁻² day⁻¹ and its seasonal amount was equivalent to 0.23% of plant N released as N₂O. A N₂O emission coefficient (N₂O_E, mg N₂O-N g⁻¹ C day⁻¹), defined as N₂O-N emission in milligrams from per gram carbon of plant dry matter within a day, was represented by a 5-fold variation ranging from 0.021 to 0.004 mg N₂O-N g C⁻¹ day⁻¹. A linear relationship (y=0.4611x+0.0015, r ²=0.9352, p < 0.001) between N₂O_E (y) and plant dark respiration rate (x, mg CO₂-C g C⁻¹ day⁻¹) suggested that in the absence of photosynthesis, some N₂O production in plant N assimilation was associated with plant respiration. Although this study could not show whether N₂O was produced or transferred by winter wheat plants, these results indicated an important role for higher plant in N₂O exchange. Identifying its potential contribution is critical for understanding agricultural ecosystem N₂O sources.     

Metabolism of 2-oxoacid analogues of leucine,valine and phenylalanine by heart muscle,brain and kidney of the rat

[1-¹⁴C]-Labelled 2-oxoacid analogues of leucine, valine and phenylalanine were used to study the metabolism of these 2-oxoacids in the brain, kidney and heart muscle of rats. By following the ¹⁴CO₂ release during 30–60 min of incubation at 37°C the decarboxylation rate was determined and measurement of the ¹⁴C-incorporation into the corresponding amino acid yielded the transamination rate. From these rates, decarboxylation/transamination ratios could be calculated which are indicative for the metabolic fate of the 2-oxoacid in the various organs. The results obtained show that all three tissues are capable of utilizing the 2-oxoacid analogues of leucine, valine and phenylalanine, however, to a different extent: kidney > heart muscle > brain. The decarboxylation/transamination ratios reveal that the branched-chain 2-oxoacids are predominantly decarboxylated in kidney and heart muscle while in brain they are mainly transaminated. The ratios calculated for phenylpyruvate in all tissues are within 0.19 and 0.36, indicating that this 2-oxoacid is preferentially transaminated. The results are discussed with respect to possible dietary alterations of enzymes involved in 2-oxoacid metabolism in order to improve transamination of these compounds.