Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2, 3-butanediol (K(m) = 14 mm, k(cat) = 78,000 min(-)(1)) and meso-butanediol (K(m) = 65 mm, k(cat) = 46,000 min(-)(1)) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K(m) = 4.5 mm, k(cat) = 98,000 min(-)(1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3. 5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R, 3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.     

Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains.

The substrate oxidation profiles of Sphingomonas yanoikuyae B1 biphenyl-2,3-dioxygenase and cis-biphenyl dihydrodiol dehydrogenase activities were examined with 1,2-dihydronaphthalene and various cis-diols as substrates. m-Xylene-induced cells of strain B1 oxidized 1,2-dihydronaphthalene to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2-3,4-tetrahydronaphthalene as the major product (73% relative yield). Small amounts of (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (15%), naphthalene (6%), and alpha-tetralone (6%) were also formed. Strain B8/36, which lacks an active cis-biphenyl dihydrodiol dehydrogenase, formed (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene (51%), in addition to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (44%) and (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (5%). The cis-biphenyl dihydrodiol dehydrogenase of strain B1 oxidized both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene, but only the (+)-(1S,2R)-enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene. The results show that biphenyl dioxygenase expressed by S. yanoikuyae catalyzes dioxygenation, monooxygenation, and desaturation reactions with 1,2-dihydronaphthalene as the substrate, and cis-biphenyl dihydrodiol dehydrogenase catalyzes the enantioselective dehydrogenation of (+)-(1S,2R)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (+)-(1S,2R)-cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene.     

On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.     

Biotransformation of benzothiophene by isopropylbenzene-degrading bacteria.

Isopropylbenzene-degrading bacteria, including Pseudomonas putida RE204, transform benzothiophene to a mixture of compounds. Induced strain RE204 and a number of its Tn5 mutant derivatives were used to accumulate these compounds and their precursors from benzothiophene. These metabolites were subsequently identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. When strain RE204 was incubated with benzothiophene, it produced a bright yellow compound, identified as trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate, formed by the rearrangement of cis-4-(3-keto-2,3-dihydrothienyl)-2-hydroxybuta-2,4-dieno ate, the product of 3-isopropylcatechol-2,3-dioxygenase-catalyzed ring cleavage of 4,5-dihydroxybenzothiophene, as well as 2-mercaptophenylglyoxalate and 2'-mercaptomandelaldehyde. A dihydrodiol dehydrogenase-deficient mutant, strain RE213, converted benzothiophene to cis-4,5-dihydroxy-4,5-dihydrobenzothiophene and 2'-mercaptomandelaldehyde; neither trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate nor 2-mercaptophenylglyoxalate was detected. Cell extracts of strain RE204 catalyzed the conversion of cis-4,5-dihydroxy-4,5-dihydrobenzothiophene to trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate in the presence of NAD+. Under the same conditions, extracts of the 3-isopropylcatechol-2,3-dioxygenase-deficient mutant RE215 acted on cis-4,5-dihydroxy-4,5-dihydrobenzothiophene, forming 4,5-dihydroxybenzothiophene. These data indicate that oxidation of benzothiophene by strain RE204 is initiated at either ring. Transformation initiated at the 4,5 position on the benzene ring proceeds by three enzyme-catalyzed reactions through ring cleavage. The sequence of events that occurs following attack at the 2,3 position of the thiophene ring is less clear, but it is proposed that 2,3 dioxygenation yields a product that is both a cis-dihydrodiol and a thiohemiacetal, which as a result of this structure undergoes two competing reactions: either spontaneous opening of the ring, yielding 2'-mercaptomandelaldehyde, or oxidation by the dihydrodiol dehydrogenase to another thiohemiacetal, 2-hydroxy-3-oxo-2,3-dihydrobenzothiophene, which is not a substrate for the ring cleavage dioxygenase but which spontaneously opens to form 2-mercaptophenylglyoxaldehyde and subsequently 2-mercaptophenylglyoxalate. The yellow product, trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate, is a structural analog of trans-o-hydroxybenzylidenepyruvate, an intermediate of the naphthalene catabolic pathway; extracts of recombinant bacteria containing trans-o-hydroxybenzylidenepyruvate hydratase-aldolase catalyzed the conversion of trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate to 3-hydroxythiophene-2-carboxaldehyde, which could then be further acted on, in the presence of NAD+, by extracts of recombinant bacteria containing the subsequent enzyme of the naphthalene pathway, salicylaldehyde dehydrogenase.     

Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.     

Assay of physiological levels of 2,3-butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry

We present an assay for 2,3-butanediol by gas chromatography-mass spectrometry of its trimethylsilyl ethers. 2R,3R- and/or 2S,3S-2,3-butanediol and meso-2,3-butanediol are quantitated with corresponding internal standards of [2,3-2H2]butanediol. Limits of detection are 1 and 0.1 microM for split and splitless injections, respectively. Blood concentrations of 2,3-butanediol in nonalcoholics are 0.5 +/- 0.3 (SD) microM for 2R,3R- and/or 2S,3S-2,3-butanediol and 0.8 +/- 0.4 microM for meso-2,3-butanediol (n = 9). Two hours after alcohol ingestion, blood levels had risen in eight of nine subjects to 1.2 +/- 0.7 microM for 2R,3R-/2S,3S-2,3-butanediol and to 1.2 +/- 0.6 microM for meso-2,3-butanediol. Baseline urinary excretion of 2,3-butanediol is 0.4 +/- 0.2 mumol/mmol creatinine for 2R,3R-/2S,3S-2,3-butanediol and 0.9 +/- 0.5 mumol/mmol creatinine for meso-2,3-butanediol.     

Biodegradation of Dibenzo-p-dioxin, Dibenzofuran, and Chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03

The dioxin-degrading strain Pseudomonas veronii PH-03 was isolated from contaminated soil by selective enrichment techniques. Strain PH-03 grew on dibenzo-p-dioxin and dibenzofuran as a sole carbon source. Further, 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxin and other dioxin metabolites, salicylic acid, and catechol were also metabolized well. Resting cells of strain PH-03 transformed dibenzo-p-dioxin, dibenzofuran, 2,2',3-trihydroxybiphenyl, and some chlorodioxins to their corresponding metabolic intermediates such as catechol, salicylic acid, 2-hydroxy-(2-hydroxyphenoxy)-6-oxo-2,4-hexadienoic acid, and chlorocatechols. The formation of these metabolites was confirmed by comparison of gas chromatography-mass spectrometry (GC-MS) data with those of authentic compounds. Although we did observe the production of 3,4,5,6-tetrachlorocatechol (3,4,5,6-TECC) from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) with resting cell suspensions of PH-03, growth of strain PH-03 in the presence of 1,2,3,4-TCDD was poor. This result suggests that strain PH-03 is unable to utilize 3,4,5,6-TECC, even at very low concentration (0.01 mM) due to its toxicity. In cell-free extracts of DF-grown cells, 2,2',3-trihydroxybiphenyl dioxygenase, 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase, and catechol-2,3-dioxygense activities were detected. Moreover, the activities of meta-pyrocatechase and 2,2',3-trihydroxybiphenyl dioxygenase from the crude cell-free extracts were inhibited by 3-chlorocatechol. However, no inhibition was observed in intact cells when 3-chlorocatechol was formed as intermediate.     

l-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine

The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.     

Production of a chiral alcohol, 1-(3,4-dihydroxyphenyl) ethanol, by mushroom tyrosinase

1-(3,4-Dihydroxyphenyl) ethanol was produced biocatalytically for the first time using mushroom tyrosinase. 4-Ethylphenol at 1 mM was consumed over 12 min giving 0.23 mM 4-ethylcatechol and 0.36 mM (R/S)-1-(3,4-dihydroxyphenyl) ethanol (ee 0.5 %). Mushroom tyrosinase consumed 4-ethylphenol at 6.7 μmol min^?1 mg protein^?1 while the rates of formation of 4-ethylcatechol and 1-(3,4-dihydroxyphenyl) ethanol were 1.1 and 1.9 μmol min^?1 mg protein^?1. Addition of the ascorbic acid, as a reducing agent to biotransformation reactions, increased 4-ethylcatechol formation by 340 %. However, accumulation of 1-(3,4-dihydroxyphenyl) ethanol was not observed in the presence of ascorbic acid. While the 1-(3,4-dihydroxyphenyl) ethanol was racemic, it is the first chiral product produced by tyrosinase starting from a non-chiral substrate.     

Isolation and Characterization of a Dibenzofuran-Degrading Yeast: Identification of Oxidation and Ring Cleavage Products

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2,3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[b]furanylidene glycolic acid, which were characterized by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.     

The temperature dependence of steady-state kinetics: What can be learned about pig liver esterase stereospecificity?

The steady-state kinetic parameters for pig liver carboxylesterase (PLE)-catalyzed hydrolysis of the prochiral substrate dimethyl phenylmalonate (DMPM) (product enantioselectivity) and the separate enantiomers of three chiral 2-phenylpropionic acid esters (substrate enantioselectivity) were measured at seven temperatures between 288 K and 312 K. Arrhenius plots of turnover numbers against the reciprocal of experimental temperatures yielded enthalpies and entropies of activation at enzyme saturation. (+)-(S)-methyl-2-phenylpropionate, (+)-(S)-4-nitrophenyl 2-phenylpropionate, and both enantiomers of phenyl 2-phenylpropionate showed very similar activation enthalpies and entropies (approximately 50 kJ mol^?1 and ?50 J mol^?1 K^?1, respectively), but differences were observed for (?)-(R)-methyl 2-phenylpropionate and (?)-(R)-4-nitrophenyl 2-phenylpropionate. Whereas the entropies of activation of all 2-phenylpropionates were negative, positive entropies of activation were measured in the formation of monomethyl phenylmalonate enantiomers from DMPM. Enthalpy–entropy compensation analysis of the data indicates a common mechanism of PLE substrate and product enantiospecificity in the reactions studied here. © 1994 Wiley-Liss, Inc.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus

According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser-desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser-desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3-hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3-hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A.     

Enantio- and regioselectivity in the epoxide-hydrolase-catalyzed ring opening of aliphatic oxiranes: Part II: Dialkyl- and trialkylsubstituted oxiranes.

The extent of substrate enantioselectivity and regioselectivity of a series of aliphatic 2,3-dialkyl- and trialkylsubstituted oxiranes in their in vitro epoxide-hydrolase-catalyzed hydrolysis depends on the size of the alkyl residues and on the substitution pattern of the oxirane ring. The enzyme-catalyzed hydrolysis of cis-oxiranes, containing at least one methyl substituent, shows complete or nearly complete substrate enantioselectivity and regioselectivity with nucleophilic attack by water occurring with inversion of configuration at the methylsubstituted ring carbon atom of (S)-configuration. In the hydrolysis of the isomeric trans-oxiranes, both enantiomers are metabolized with a higher rate for the (2S;3S)-enantiomer. The conversion of trimethyloxirane occurs with high substrate enantioselectivity in favor of the (S)-enantiomer and with complete regioselectivity at the monomethylsubstituted ring carbon atom. The differentiation of the enantiotopic ring carbon atoms (product enantioselectivity) in the smallest aliphatic meso-oxirane, cis-2,3-dimethyloxirane, leads to (2R;3R)-butane-2,3-diol with ee = 86%. cis-2-Ethyl-3-propyloxirane, possessing alkyl residues larger than methyl, represents an extremely poor substrate in the epoxide-hydrolase-catalyzed hydrolysis process.     

Deglycosylation of puerarin and other aromatic C-glucosides by a newly isolated human intestinal bacterium

The human intestinal microbiota may influence the fate of bioactive polyphenols, such as the isoflavone puerarin (daidzein 8-C-glucoside), following their oral intake. Faecal suspensions from 19 healthy subjects were tested for their ability to C-deglycosylate puerarin. Only one of these catalysed this reaction. A rod-shaped Gram-positive bacterium, strain CG19-1, capable of deglycosylating puerarin to daidzein was isolated from the corresponding suspension. However, the strictly anaerobic isolate was unable to utilize puerarin as sole carbon and energy source nor any of the tested carbohydrates. Comparative 16S rRNA gene sequence analysis indicated that strain CG19-1 is a new species of the Lachnospiraceae. Strain CG19-1 also converted other aromatic C-glucosides in addition to puerarin. The xanthone C-glucoside mangiferin was deglycosylated to norathyriol. The flavone C-glucosides homoorientin and vitexin were degraded to 3-(3,4-dihydroxyphenyl)propionic acid via luteolin and 3-(4-hydroxyphenyl)propionic acid respectively. In addition, strain CG19-1 converted flavonoid O-glucosides, but at rates that were lower than those of the C-glucosides tested. The isolate deglycosylated the isoflavone O-glucosides daidzin and genistin to daidzein and genistein respectively. Several O-glucosides of the flavones luteolin and apigenin undergoing deglycosylation were subsequently cleaved to 3-(3,4-dihydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)propionic acid respectively. Moreover, strain CG19-1 cleaved both O-desmethylangolensin and 6'-hydroxy-O-desmethylangolensin to yield 2-(4-dihydroxyphenyl)propionic acid. The corresponding cleavage product, resorcinol, was only observed for O-desmethylangolensin.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury

Previous work has shown that, shortly after rabbit corneas are injured, arachidonic acid metabolism is activated, and 12-hydroxyeicosatetraenoic acid (12-HETE) is one of the main products formed (Bazan, H. E. P., Birkle, D. L., Beuerman, R., and Bazan, N. G. (1985) Invest. Ophthalmol. & Visual Sci. 26, 474-480; Bazan, H. E. P. (1987) Invest. Ophthalmol. Visual Sci. 28, 314-319). In order to determine whether this metabolite is a lipoxygenase product, anesthetized rabbit corneas injured in vivo, either cryogenically or by 1 M NaOH, were subsequently incubated in vitro with [14C] arachidonic acid in the presence of indomethacin. 12-HETE was the main metabolite produced, as established by gas chromatography-mass spectrometry. The (R)- and (S)-enantiomers of novel naphthoyl-pentafluorobenzoyl derivatives of 12-HETE were resolved by chiral-phase high performance liquid chromatography. The radiolabeled 12-HETE from whole cornea and from isolated epithelium, endothelium, or stroma eluted as a single peak co-chromatographing with the (S)-enantiomer and was detected both by UV absorbance at 234 nm and by radioactivity. In noninjured corneas a smaller peak of radiolabeled (12S)-HETE was also eluted from the chiral column. The stereochemistry was additionally confirmed by liquid chromatography-mass spectrometry. These studies suggest that (12S)-lipoxygenase is activated in the injured rabbit cornea.