The formation of shikimate-3-phosphate in cell-free preparations of Sorghum

Shikimate-3-phosphate was formed by cell-free preparations of etiolated shoots and green stems of Sorghum bicolor but not of roots of etiolated see     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

The reduction of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one by thiols

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a naturally occurring hydroxamic acid involved in pest resistance of cereals, was reduced by thiols to the corresponding lactam. Kinetic studies showed that the reactive species are undissociated DIMBOA and thiolate anion. Possible mechanisms for the reaction are discussed in the light of relative reactivities of DIMBOA and a compound lacking the 7-methoxy substituent, and results from molecular orbital calculations.     

3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.     

The Effect of Gamma-Irradiation on the Sucrose Content in Sweet Potato Roots and Potato Tubers

The sucrose content in both potato tubers and sweet potato roots was considerably increased by gamma-irradiation. The maximum increase was achieved by an irradiation dose of 3 to 4 kGy for potatoes and 0.8 to 2 kGy for sweet potatoes. Cooling treatment (15°C, 2 weeks) for sweet potato roots also enhanced the sucrose content (almost 2 times) but was not additive to the irradiation treatment; the maximum sucrose content in irradiated sweet potato roots was in the range of 7 to 12% irrespective of the cooling treatment, depending on the variety of sweet potatoes. Irradiation made the sucrose content in the roots 2 to 4 times higher.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Synthesis of 5-aminolevulinic acid-[14C] by cell-free preparations from greening maize leaves

The conversion of 2-ketoglutarate-[¹⁴C] to 5-aminolevulinic acid-[¹⁴C] (ALA) by a cell-free system from maize leaves is described. Optimal conversion was achieved at pH 6.2 using a 20 000 g supernatant after gel filtration through Sephadex G-25. The formation of ALA required Mg²⁺, an amino donor (alanine or glutamate), pyridoxal phosphate and NADH. NADPH was somewhat less effective.     

Indol-3yl-acetic acid in roots of Zea mays

Indol-3yl-acetic acid was identified in extracts of sterile roots of Zeamays seedlings by means of TLC, chromogenic reactions, GLC and GC-MS.     

Degradation of diazinon by 2,4-dihydroxy-7-methoxy-2h-1, 4-benzoxazin-3(4h)-one in maize

A cyclic hydroxamate, 2,4-dihydroxy-7-methoxy-2H- 1,4-benzoxazin-3(4H)-one (DIMBOA), was isolated and identified from shoots of 6-day-old corn seedlings grown in the dark. From 100 g of plant tissue 100 mg of DIMBOA were isolated. This hydroxamate was very effective in catalysing the hydrolysis of the pyrimidinyl organophosphate insecticide, diazinon (O, O-diethyl- O-[6- methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) to 6- methyl-2-(1-methylethyl)-4-hydroxypyrimidine and diethyl phosphorothioic acid. The optimum pH for hydrolytic activity was 5 and at pH values equal to or higher than the pK_a of the hydroxamic group (6.95) most of the activity was lost.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Differential sensitivity of bacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicide glyphosate

Abstract The potent inhibition of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase by the broad-spectrum herbicide glyphosate ( N -[phosphonomethyl]glycine) was confirmed for the enzymes extracted from various bacteria, a green alga and higher plants. However, 5 out of 6 species belonging to the genus Pseudomonas were found to have EPSP synthases with a 50- to 100-fold decreased sensitivity to the inhibitor. Correspondingly, growth of these 5 species was not inhibited by 5 mM glyphosate, and the organisms did not excrete shikimate-3-phosphate in the presence of the herbicide.     

Functional analysis of the essential bifunctional tobacco enzyme 3-dehydroquinate dehydratase/shikimate dehydrogenase in transgenic tobacco plants

In plants, the shikimate pathway occurs in the plastid and leads to the biosynthesis of aromatic amino acids. The bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SHD) catalyses the conversion of dehydroquinate into shikimate. Expression of NtDHD/SHD was suppressed by RNAi in transgenic tobacco plants. Transgenic lines with <40% of wild-type activity displayed severe growth retardation and reduced content of aromatic amino acids and downstream products such as cholorogenic acid and lignin. Dehydroquinate, the substrate of the enzyme, accumulated. However, unexpectedly, so did the product, shikimate. To exclude that this finding is due to developmental differences between wild-type and transgenic plants, the RNAi approach was additionally carried out using a chemically inducible promoter. This approach revealed that the accumulation of shikimate was a direct effect of the reduced activity of NtDHD/SHD with a gradual accumulation of both dehydroquinate and shikimate following induction of gene silencing. As an explanation for these findings the existence of a parallel extra-plastidic shikimate pathway into which dehydroquinate is diverted is proposed. Consistent with this notion was the identification of a second DHD/SHD gene in tobacco (NtDHD/SHD-2) that lacked a plastidic targeting sequence. Expression of an NtDHD/SHD-2-GFP fusion revealed that the NtDHD/SHD-2 protein is exclusively cytosolic and is capable of shikimate biosynthesis. However, given the fact that this cytosolic shikimate synthesis cannot complement loss of the plastidial pathway it appears likely that the role of the cytosolic DHD/SHD in vivo is different from that of the plastidial enzyme. These data are discussed in the context of current models of plant intermediary metabolism.     

(+)-5-Hydroxy-dioxindole-3-acetic acid,a synergist from rice bran of auxin-induced ethylene production in plant tissue

(+)-5-Hydroxy-dioxindole-3-acetic acid (1) was isolated from rice bran as a substance synergistic with auxin in the auxin induced ethylene production by etiolated mungbean hypocotyl segments. 5-Hydroxy-oxindole-3-acetic acid (4) and IAA were also obtained. The importance of a hydroxyl group in the 5-position in the two compounds was suggested since synthesized (±)-dioxindole-3-acetic acid (6) was inactive.     

The biosynthesis of melanin in Alternaria

A melanin which is insoluble in strong alkali has been isolated from Alternaria mycelium. Alkali fusion of the pigment produced p-hydroxybenzoa     

Increase of auxin-antagonistic action in 3-phosphoryloxycarbostyrils

Introduction of phosphoryloxy group at C-3 in carbostyrils has been found to increase auxin-antagonistic action. 3-Phosphoryloxycarbostyrils were synthesised from 3-hydroxycarbostyril and 3-hydroxy-4-phenylcarbostyril (viridicatin). The phosphates at very low concentrations markedly stimulated the root growth of rice seedlings, and competitively inhibited the IAA-induced lamina inclination in rice explants.     

Inhibition by isonicotinyl hydrazide of pigment formation in higher plants

The inhibition of greening of illuminated etiolated maize seedlings by isonicotinyl hydrazide can be alleviated by serine or pyruvate. The similar inhibition in barley can be reversed only by pyruvate. In both plants earlier intermediates in the glycollate pathway and other related compounds were ineffective in overcoming the inhibition of greening produced by isonicotinyl hydrazide. In maize seedlings radioactivity from l-serine-[3-¹⁴C] is poorly incorporated into β-carotene, a typical chloroplast terpenoid, unless glycine and formate or, more effectively, glycine together with isonicotinyl hydrazide are supplied. These supplementations may minimize interconversion of serine and glycine, and hence dilution of radioactivity at C-3 of l-serine by unlabelled C-1 units, before incorporation into terpenoids. The results support the view that in young greening tissue the C2-3 fragment of l-serine can give rise to acetyl-CoA, an obligatory precursor of chloroplast terpenoids.     

Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) from shoot tissue of pea seedlings was purified to apparent homogeneity by sequential ammonium-sulphate precipitation, ion-exchange and hydrophobic-interaction chromatography and substrate elution from cellulose phosphate. Gel electrophoresis and gel-permeation chromatography showed that the purified enzyme was monomeric with molecular weight 50,000. The herbicide glyphosate was a potent inhibitor of the forward enzyme-catalyzed reaction.Abbreviations DEAE diethylaminoethyl - EPSP 5-enolpyruvylshikimate 3-phosphate     

生物法合成3-羟基丙酸的研究进展

从3-羟基丙酸的性质出发,介绍了生物法合成3-羟基丙酸以及它在生物体内的五种代谢途径,此外还简要介绍了3-羟基丙酸在合成生物聚酯、抗植物病虫害上的一些应用。