A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity

A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.     

Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona`Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.     

Purification and characterization of D-glycerate-3-kinase from maize leaves

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min^-1 · (mg protein) ^-1. The enzyme was a monomer of a relative molecular mass (M_r) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K _ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K _i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K _is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C₄-photosynthesis.Abbreviations DEAE diethylaminoethyl - kDa kdalton - GAP-DH glyceraldehyde phosphate dehydrogenase - GK glycerate kinase - LDH lactate dehydrogenase - 2-ME 2-mercaptoethanol - M_r relative molecular mass - PEP phosphoenolpyruvate - PGA(PK) phosphoglycerate (phosphokinase) - PK pyruvate kinase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Glycosides of 2-C-methyl-D-erythritol from the fruits of anise,coriander and cumin

Eight glycosides of 2-C-methyl-D-erythritol (1) were isolated from the fruit of anise, and their structures were clarified as 1-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranoside, 4-O-beta-D-glucopyranoside, 1-O-beta-D-fructofuranoside, 3-O-beta-D-fructofuranoside, 4-O-beta-D-fructofuranoside, 1-O-beta-D-(6-O-4-hydroxybenzoyl)-glucopyranoside and 1-O-beta-D-(6-O-4-methoxybenzoyl)-glucopyranoside of 2-C-methyl-D-erythritol (2-9), respectively. Furthermore, 2 and 4 were isolated from the fruit of coriander, and 2, 3 and 4 were isolated from the fruit of cumin. Though the phosphate of 1 was known to be one of the first precursors of isoprenoids in the non-mevalonate pathway, and 1 is considered to be a common constituent in Umbelliferous plants, the glycosides of 1 are found for the first time.     

Biosynthesis of anthraquinones in cell cultures of Cinchona 'Robusta' proceeds via the methylerythritol 4-phosphate pathway.

Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate.     

A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase

The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.     

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.     

2-C-methyl-D-erythritol is a major carbohydrate in petals of Phlox subulata possibly involved in flower development

2-C-methyl-D-erythritol, a soluble carbohydrate that is not ubiquitously found in higher plants, was detected in the ethanol extract from Phlox subulata petals and isolated using HPLC. The isolated compound was identified by 1H-NMR, 13C-NMR and Cl-MS spectra. 2-C-methyl-D-erythritol was a major soluble carbohydrate in petals, leaves and stems. In petals, the concentration of 2-C-methyl-D-erythritol markedly increased during flower development and opening and was similar in concentration to glucose, a ubiquitous metabolic sugar. This suggests that 2-C-methyl-D-erythritol may contribute to flower opening in association with glucose in the P. subulata.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Crystal structure of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase,an enzyme in the non-mevalonate pathway of isoprenoid synthesis

The crystal structure of the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) kinase from the thermophilic bacterium Thermus thermophilus HB8 has been determined at 1.7-A resolution. This enzyme catalyzes phosphorylation of the 2-hydroxyl group of CDP-ME, the fourth step of the non-mevalonate pathway, which is essential for isoprenoid biosynthesis in several pathogenic microorganisms. Since this pathway is absent in humans, it is an important target for the development of novel antimicrobial compounds. The structure of the enzyme is similar to the structures of mevalonate kinase and homoserine kinase, members of the GHMP superfamily. Lys8 and Asp125 are active site residues in mevalonate kinase that also appear to play a catalytic role in CDP-ME kinase. Both the mevalonate and the non-mevalonate pathways therefore involve closely related kinases with similar mechanisms. Assaying the enzyme showed that CDP-ME kinase will phosphorylate CDP-ME but not 4-(uridine 5'-diphospho)-2-C-methyl-D-erythritol, indicating the substrate pyrimidine moiety is involved in important interactions with the enzyme.     

Biochemical characterization of human and murine isoforms of UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine 2-epimerase/<Emphasis Type="Italic">N</Emphasis>-acetylmannosamine kinase (GNE)

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.     

The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.     

Characterization of a Sorghum bicolor gene family encoding putative protein kinases with a high similarity to the yeast SNF1 protein kinase

C₄ photosynthesis is functionally dependent on metabolic interactions between mesophyll and bundle-sheath cells. Although the C₄ cycle is biochemically well understood many aspects of the regulation of enzyme activities, gene expression and cell differentiation are elusive.Protein kinases are likely involved in these regulatory processes providing links to hormonal, metabolic and developmental signal transduction pathways. We have identified several protein kinases that are differentially expressed in mesophyll and bundle-sheath cells of the C₄ plant Sorghum bicolor. Here we describe the characterization of two putative protein kinases that show high similarity to the SNF1/AMPK family of protein serine/threonine kinases. The mRNA of both kinases accumulates to much higher levels in mesophyll cells than in the bundle-sheath and can also be detected in root tissue. Complementation experiments with a snf1 mutant of Saccharomyces cerevisiae indicate that the S. bicolor protein kinase SNFL1 does not represent a functional homologue of the yeast SNF1 protein kinase.     

Purification and characterization of glycerate kinase from the thermoacidophilic archaeonThermoplasma acidophilum: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withK _m values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ca²⁺, and Sr²⁺, were substituted for Mg²⁺, the enzyme activities were less than 10% of that obtained in the presence of Mg²⁺. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.     

Immunochemical Approach to the Problem of Differential Determination of Natural Forms of Abscisic Acid

An original modification of the standard ELISA procedure for differential determination of different forms of abscisic acid (ABA) is proposed. It is shown that endogenous forms of ABA may be quantitatively determined in plant tissues subjected to minimal treatment, without purification of the hormones and their chemical modification. The modification has been approved when analyzing changes in the content of different ABA forms in plant tissues differing in physiological activity. Quantitative differential determination of changes in the content of different ABA forms has been performed in ovaries of Triticum aestivum L. and Taraxacum officinale Web. in the period of activity of the ovule (from the moment of its activation to the beginning of division). It is shown that, despite the different types of reproduction in the species studied (amphimixis and apomixis), the time course of changes in the content of different forms of ABA in ovaries is similar, which is suggestive of a correlation between the activity of endogenous hormonal system and chronology of main events (e.g., the beginning of endospermogenesis) of the reproductive cycle.     

Gas chromatographic–mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples

A simple and rapid method is described for the GC–MS determination of 4-nonylphenols (NOs) and 4-tert-octylphenol (OC) in biological samples. The NOs and OC in the sample are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR column clean-up, the sample extract is analyzed by GC–MS in the selected ion monitoring (SIM) mode. Average recoveries in pale chub (fish) and corbicula (shellfish) are 86.0 and 93.4% for NOs, and 95.8 and 96.4% for OC, respectively, spiked at the levels of 1.0 μg of NOs and 0.1 μg of OC per 5 g of fish and shellfish samples. The detection limits are 20 ng/g for NOs and 2 ng/g for OC.     

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.     

Genetic evidence for functional specificity of the yeast GCN2 kinase

In yeast the GCN2 kinase mediates translational control ofGCN4 by phosphorylating the subunit of eIF-2 in response to extracellular amino acid limitation. Although phosphorylation of eIF-2 has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect onGCN4 mRNA translation. Under the same conditions, translation of other mRNAs appears only slightly affected. The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear. Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitatesGCN4-specific translational regulation.     

腺苷酸激酶基因在大肠杆菌中的可溶性高表达

报道了鸡肌腺苷酸激酶基因的克隆和在温控启动子P_RP_L调控下在大肠杆菌中的可溶性高效表达.SDS-PAGE分析表明,鸡肌腺苷酸激酶的含量可占大肠杆菌细胞总蛋白含量的38％.利用Johnson等的干冰/乙醇-冰水浴反复冻融法,可将此重组蛋白进行富集,纯度可达85％以上.鸡肌腺苷酸激酶可与抗兔肌腺苷酸激酶单克隆抗体产生强的交叉反应.