Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400

Abstract In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho -dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.     

Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.     

Derivatives of 2,3-anhydro-dl-threitol, 2,3:4,5-dianhydrogalactitiol,and 2,3:4,5-dianhydroallitol

α,ω-Disubstituted derivatives of 2,3-anhydro-dl-threitol (2), 2,3-anhydro-erythritol (4), 2,3:4,5-dianhydrogalactitol (8), and 2.3:4,5-dianhydroallitol (12) have been synthesised by epoxidation of the appropriate alkenes and dienes. Benzyloxycarbonyl groups were used for protecting the primary hydroxyl groups during epoxidation.     

Preparation of 2,3-seco-5 alpha-cholestane-2,3-diol and 4 alpha-methyl-2,3-seco-5 alpha-cholestane-2,3-diol and its reactions with o-nitrophenyl selenocyanate

The reaction of 2,3-seco-5 alpha-cholestane-2,3-diol and 4 alpha-methyl-2,3-seco-5 alpha-cholestane-2,3-diol with o-nitrophenyl selenocyanate was studied. The diols were synthesized from cholesterol.     

Biological production of 2,3-butanediol

2,3-Butanediol (2,3-BDL), which is very important for a variety of chemical feedstocks and liquid fuels, can be derived from the bioconversion of natural resources. One of its well known applications is the formation of methyl ethyl ketone, by dehydration, which can be used as a liquid fuel additive. This article briefly reviews the basic properties of 2,3-BDL and the metabolic pathway for the microbial formation of 2,3-BDL. Both the biological production of 2,3-BDL and the variety of strains being used are introduced. Genetically improved strains for BDL production which follow either the original mechanisms or new mechanisms are also described. Studies on fermentation conditions are briefly reviewed. On-line analysis, modeling, and control of BDL fermentation are discussed. In addition, downstream recovery of 2,3-BDL and the integrated process (being important issues of BDL production) are also introduced.     

Acid-induced fusion of liposomes: studies with 2,3-seco-5 alpha-cholestan-2,3-dioic acid

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.     

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.     

1-Arylmethyl-2,3-dioxo-2,3-dihydroindole thiosemicarbazones as leads for developing cytotoxins and anticonvulsants

Various substituted 1-arylmethyl-2,3-dioxo-2,3-dihydroindole thiosemicarbazones 3a-h, 1-benzyl-2,3-dioxo-2,3-dihydroindole N(4)-aryl thiosemicarbazones 4a-i and 1-benzyl-2,3-dioxy-2,3-dihydroindole N(4)-cyclohexylthiocarbazone 5 were synthesized. All of these compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 leukemia cells. Nearly 40% of these compounds possess low micromolar IC(50) values and some are either more potent than, or equipotent with, melphalan. Various correlations between the structures of these compounds and cytotoxic potencies were obtained which included the use of QSAR and molecular modeling techniques. Representative compounds displayed anticonvulsant properties in rats and were well tolerated by these animals. The encouraging biodata noted affords adequate rationale for outlining guidelines for further development of these molecular scaffolds.     

pH-dependent changes of 2,3-bisphosphoglycerate.

A systematic study of the pH dependent changes in the range 6.6--7.4 of 2,3 bisphosphoglycerate (2,3-DPG) was performed in the presence and absence of glucose during transitional and steady states. The results indicate that 2,3-DPGase breaks down 2,3-DPG nealy independent of pH at a rate of 480 mu moles 2,3-DPG/1 cells.h. The 2,3-DPG mutase is practically completely inhibited below pH 6.9. The 2,3-DPG level in the presence of glucose reaches a pH dependent steady state after about 18 h. The share of the 2,3-DPG bypass in the steady state decreases from 24% at pH 7.4 to 12% at pH 7.0. The formation of pyruvate corresponds to the beadkdown of 2,3-DPG after consumption of an unknown reducing substance.     

Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erytrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC 2.7.5.4/EC 3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.     

Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.     

The potential antioxidant activity of 2,3-dihydroselenophene, a prototype drug of 4-aryl-2,3-dihydroselenophenes

Here we present our results in palladium cross-coupling reaction of aryl boronic acids with 4-iodo-2,3-dihydroselenophene derivatives. The cross-coupled products were obtained in satisfactory yields. A dehydrogenation of 4,5-diphenyl-2,3-dihydroselenophene was activated by DDQ and the 2,3-diarylselenophene was obtained in good yield. Regarding the antioxidant activity, the selenophene derivative 3a was effective in counteracting lipid and protein oxidation as well as scavenging ABTS radical. The findings of the present study indicate that 3a is a prototype for future drug development programs to treat disorders mediated by reactive oxygen species.     

Nicotinic acid metabolism. 2,3-Dimethylmalate lyase

1) A new enzyme, 2,3-dimethylmalate lyase, was purified from Clostridium barkeri to about 80% homogeneity. Some of the properties of the enzyme are described. 2) It is shown that the 2,3-dimethylmalic acid (m.p. 143 degrees C) described in the literature represents only one racemic pair. This pair is not attacked by 2,3-dimethylmalate lyase. 3) The isolation of both racemic pairs of 2,3-dimethylmalic acid is described. Half of one pair, m.p. 104-106 degrees C, was converted to propionate and pyruvate by 2,3-dimethylmalate lyase. 4) In combination with earlier work performed by E.R. Stadtman and coworkers the results given under points 1--3 establish 2,3-dimethylmalate as an intermediate in the degradation of nicotinic acid by C. barkeri. 5) Experimental evidence indicates the 2,3-dimethylmalate lyase is no acyl-S-enzyme and that it is different in this respect as well as in quaternary structure from the apparently related enzymes citrate lyase and citramalate lyase.     

Mechanistic studies on trans-2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (Ent A) in the biosynthesis of the iron chelator enterobactin

The enzyme 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (2,3-diDHB dehydrogenase, hereafter Ent A), the product of the enterobactin biosynthetic gene entA, catalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic product 2,3-dihydroxybenzoate (2,3-DHB). The catechol 2,3-DHB is one of the key siderophore units of enterobactin, a potent iron chelator secreted by Escherichia coli. To probe the reaction mechanism of this oxidation, a variety of 2,3-diDHB analogues were synthesized and tested as substrates. Specifically, we set out to elucidate both the regio- and stereospecificity of alcohol oxidation as well as the stereochemistry of NAD+ reduction. Of those analogues tested, only those with a C3-hydroxyl group (but not a C2-hydroxyl group) were oxidized to the corresponding ketone products. Reversibility of the Ent A catalyzed reaction was demonstrated with the corresponding NADH-dependent reduction of 3-ketocyclohexane- and cyclohexene-1-carboxylates but not the 2-keto compounds. These results establish that Ent A functions as an alcohol dehydrogenase to specifically oxidize the C3-hydroxyl group of 2,3-diDHB to produce the corresponding 2-hydroxy-3-oxo-4,6-cyclohexadiene-1-carboxylate (Scheme II) as a transient species that undergoes rapid aromatization to give 2,3-DHB. Stereospecificity of the C3 allylic alcohol group oxidation was confirmed to be 3R in a 1R,3R dihydro substrate, 3, and hydride transfer occurs to the si face of enzyme-bound NAD+.     

Characterization of catechol 2,3-dioxygenases.

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.     

Separation of Racemic frommeso-2,3-Butanediol

2,3-Butanediol containing less than 3% of themesoform has been obtained from samples containing up to 50% of themesoform. The diacetate was obtained by esterification with acetic anhydride in the presence of traces of sulfuric acid as a catalyst and was then purified. When the diacetate was held at 4°C, crystals of racemic 2,3-butanediol diacetate formed, and these were separated by filtration. The diacetate was then transformed back to 2,3-butanediol by transesterification with methanol in the presence of sodium methylate as a catalyst. The resulting 2,3-butanediol contained less than 3% of themesoform. For an original batch of 2,3-butanediol containing 50%dland 50%meso,this method can isolate up to 70% of the racemate content. If the original 2,3-butanediol contains too muchmesoform, racemic 2,3-butanediol diacetate does not crystallize, but 2,3-butanediol containing up to 60% of themesoform can be enriched up to 70% racemate by distillation.     

1-Arylmethyl-2,3-dioxo-2,3-dihydroindole thiosemicarbazones as leads for developing cytotoxins and anticonvulsants

Various substituted 1-arylmethyl-2,3-dioxo-2,3-dihydroindole thiosemicarbazones 3a-h, 1-benzyl-2,3-dioxo-2,3-dihydroindole N⁴-aryl thiosemicarbazones 4a-i and 1-benzyl-2,3-dioxy-2,3-dihydroindole N⁴-cyclohexylthiocarbazone 5 were synthesized. All of these compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 leukemia cells. Nearly 40% of these compounds possess low micromolar IC₅₀ values and some are either more potent than, or equipotent with, melphalan. Various correlations between the structures of these compounds and cytotoxic potencies were obtained which included the use of QSAR and molecular modeling techniques. Representative compounds displayed anticonvulsant properties in rats and were well tolerated by these animals. The encouraging biodata noted affords adequate rationale for outlining guidelines for further development of these molecular scaffolds.     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

Levels of glycerate 2,3-P2, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities in rat tissues. A method to quantify blood contamination of tissue extracts

The levels of glycerate 2,3-P2 and of 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities have been determined in isolated rat hepatocytes and adipocytes and in perfused rat tissues to discard blood contamination. The values obtained are much lower than those previously reported, ranging 0.50-40 nmol/g tissue. No relationship appears to exist between glycerate 2,3-P2 concentration and the levels of the enzymatic activities involved in glycerate 2,3-P2 metabolism. Assay of glycerate 2,3-P2 in tissue extracts constitute a very useful way to quantify blood contamination.