Biotransformation of the Major Fungal Metabolite 3,5-Dichloro- p-Anisyl Alcohol under Anaerobic Conditions and Its Role in Formation of Bis(3,5-Dichloro-4-Hydroxyphenyl)methane

Higher fungi have a widespread capacity for biosynthesis of organohalogens. Commonly occurring chloroaromatic fungal metabolites can end up in anaerobic microniches at the boundary of fungal colonies and wetland soils. The aim of this study was to investigate the environmental fate of a major fungal metabolite, 3,5-dichloro-p-anisyl alcohol, under anaerobic conditions. This compound was incubated with methanogenic sludge to study its biotransformation reactions. Initially, 3,5-dichloro-p-anisyl alcohol was readily demethylated in stoichiometric quantities to 3,5-dichloro-4-hydroxybenzyl alcohol. The demethylated product was converted further via two routes: a biotic route leading to the formation of 3,5-dichloro-4-hydroxybenzoate and 2,6-dichlorophenol, as well as an abiotic route leading to the formation of bis(3,5-dichloro-4-hydroxyphenyl)methane. In the first route, the benzyl alcohol moiety on the aromatic ring was oxidized, giving 3,5-dichloro-4-hydroxybenzoate as a transient or accumulating product, depending on the type of methanogenic sludge used. In sludge previously adapted to low-molecular-weight lignin from straw, a part of the 3,5-dichloro-4-hydroxybenzoate was decarboxylated, yielding detectable levels of 2,6-dichlorophenol. In the second route, 3,5-dichloro-4-hydroxybenzyl alcohol dimerized, leading to the formation of a tetrachlorinated bisphenolic compound, which was identified as bis(3,5-dichloro-4-hydroxyphenyl)methane. Since formation of this dimer was also observed in incubations with autoclaved sludge spiked with 3,5-dichloro-4-hydroxybenzyl alcohol, it was concluded that its formation was due to an abiotic process. However, demethylation of the fungal metabolite by biological processes was a prerequisite for dimerization. The most probable reaction mechanism leading to the formation of the tetrachlorinated dimer in the absence of oxygen is presented, and the possible environmental implications of its natural occurrence are discussed.     

Dynamics of organohalogen production by the ecologically important fungus Hypholoma fasciculare

The ecologically important white rot basidiomycete Hypholoma fasciculare was previously shown to produce large amounts of adsorbable organic halogens (AOX). The purposes of this study were to identify the time period of AOX production in relation to the primary and secondary metabolic phases of the growth cycle of the fungus, to determine the maximal specific AOX production rates and final AOX yields on the different substrates and to account for the measured AOX in identifiable compounds. The AOX production was observed to take place during the transition between the primary and secondary metabolic phases of the growth cycle of the fungus. The maximum AOX production rates ranged from 0.63 to 3.23 mg AOX per gram of dry mycelium per day and the final AOX yields ranged from 0.88 and 1.50 percent of dry weight of mycelium on five different substrates including natural woody substrates. The AOX produced by the fungus was stable in all five substrates, even after prolonged incubation periods. However, the composition of the AOX changed drastically. Initially most of the AOX was accounted for by the compound 3,5-dichloro-p-anisyl alcohol; however, after prolonged incubation this compound was largely converted into 3,5-dichloro-p-anisic acid in N-rich medium and into unidentified organohalogens in N-limited medium.     

Acetylenes and dichloroanisoles from Psathyrella scobinacea

The Et2O extract from Psathyrella scobinacea culture fluids contained three new acetylenic alcohols: deca-5,7,9-triynol, (-)hepta-4,6-diyne-2,3-diol, and (-)hept-cis 4-en-6-yne-2,3-diol; two known dichloroanisoles: 3,5-dichloro-4-methoxybenzaldehyde and 3,5-dichloro-4-methoxybenzyl alcohol; and three known acetylenic acids: octa-2,4,6-triynoic acid, dec-trans-2-ene-4,6,8-triynoic acid and its cis-isomer.     

Fate of substituted benzoates in the freshwater green alga, Chlamydomonas reinhardtii 11-32b

The removal of chlorinated, nitrated, and sulfonated benzoic acids in cultures of the unicellular green alga, Chlamydomonas reinhardtii 11-32b, was investigated, and the metabolic fate of a model compound, 4-chloro-3,5-dinitrobenzoic acid, was determined. The freshwater alga was able to remove a wide variety of benzoic compounds from the incubation medium. Chlamydomonas discriminated very specifically between the benzoic acids, indicated by the varying degrees of which the test compounds disappeared from the culture medium. Moreover, the alga was capable of transforming 4-chloro-3,5-dinitrobenzoic acid to several metabolites. A release of chloride ions was observed, and 3,5- dinitro-4-hydroxybenzoic acid was identified as a major transient product in the algal metabolism of 4-chloro-3,5-dinitrobenzoic acid.     

Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their applications

The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.     

Sudan-β-d-glucuronides and their use for the histochemical localization of β-glucuronidase activity in transgenic plants

 Synthesis of five different Sudan-β-d-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-d-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical localization of β-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the β-glucuronide results in the liberation of an insoluble Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants. Received: 9 December 1999 / Revision received: 25 January 2000 / Accepted: 26 January 2000     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Endoplasmic Reticulum (ER) Stress-Suppressive Compounds from Scrap Cultivation Beds of the Mushroom Hericium erinaceum

Four compounds were isolated from scrap cultivation beds of the mushroom, Hericium erinaceum. Compounds 1–4 were identified as methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, 2-chloro-1,3-dimethoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxybenzoate, and 4-chloro-3,5-dimethoxybenzaldehyde by an interpretation of the NMR and MS data, respectively. This is the first reported isolation of 1 from a natural source. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Metabolism of halogenated compounds in the white rot fungus Bjerkandera adusta studied by membrane inlet mass spectrometry and tandem mass spectrometry

Membrane inlet mass spectrometry has been used for the characterization of halogenated organic compounds produced by the fungus Bjerkandera adusta. Using this technique we obtained electron impact-, chemical ionization-, electron capture negative chemical ionization-mass spectra and tandem mass spectra directly from the growth medium. Through this direct analysis of the samples we identified novel bioconversion products and confirmed recently published data on the production of both chlorinated and brominated methoxybenzaldehyde metabolites. Growth profiles of the culture grown on a defined medium showed that the production of secondary metabolites starts after approximately 6 days and reaches maximal concentrations of 25-250 muM after 15-20 days. Although delayed, the production of secondary metabolites paralleled a depletion of glucose from the medium and stopped shortly after all glucose had been consumed. Experiments in which fluoro- and bromo-labeled 4-methoxybenzaldehydes were added to the medium at day 8 showed biotransformation of these compounds into chloro-3-fluoro-4-methoxy-benzaldehyde and chloro-3-bromo-4-methoxybenzaldehyde, respectively. No dichlorinated products were observed, suggesting that halogenation takes place only at the meta position on the 4-methoxybenzaldehydes. These experiments are the first to bring direct evidence of a halogenation mechanism, where the enzymatic attack takes place directly on the 4-methoxybenzaldehyde intermediates. (c) John Wiley & Sons, Inc.     

Brominated anisoles and cresols in the red alga Polysiphonia sphaerocarpa

The red marine alga Polysiphonia sphaerocarpa was extracted by a simultaneous steam distillation-solvent extraction technique and several brominated compounds were identified by gas chromatography-mass spectrometry. The compounds detected were 2,4-dibromoanisole, 2,4,6-tribromoanisole, 3-bromocresol, 3,5-dibromocresol, 3-bromo-4-hydroxybenzaldehyde, 3,5-dibromo-4-hydroxybenzaldehyde, 2-bromophenol, 4-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol and 2,4,6-tribromophenol. This is the first time brominated anisoles and cresols have been detected in marine algae.     

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. Received: 22 March 1999 / Received revision: 12 July 1999 / Accepted: 1 August 1999     

Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas fluorescens

A strain of Pseudomonas fluorescens was capable of co-metabolizing 3-chlorobenzoic acid with the production of a chlorinated catechol black pigment. A peroxidase and another enzymatic activity referred to as a polyphenol oxidase were found to be involved in the oxidation of 4-chlorocatechol to 4-chloro-1,2-benzoquinone, i.e. in the production of highly reactive substrates for pigment formation. Therefore, P. fluorescens cells were seen to take an active part not only in 3-chlorobenzoate mineralization but also in overall pigment production. pH was found to be a key parameter in the regulation of the activity of P. fluorescens oxidoreductive enzymes. Ultrastructural investigations showed that electron dense granules of pigment were distributed throughout the cytoplasm of Pseudomonas fluorescens cells grown in presence of 3-chlorobenzoate, as confirmed also by Thiéry cytochemical investigations.In these cells, an extensive contraction of the cytoplasm as well as a significant damage to the cell wall after two days of incubation, suggested that pigment production caused a premature death of the cells accompanied by the leakage of the cell content. Pigment production seemed to occur mostly in the cytoplasmic context where the electron dense material accumulates until it is released in the medium after the cell lysis.Abbreviations 3-CBA 3-chlorobenzoic acid - BA benzoic acid - 4-CC 4-chlorocatechol - 3-CC 3-chlorocatechol - MBTH 3-methyl-2-benzothiazolinone hydrazone - l-DOPA l-3,4-dihydroxyphenyl-alanine - SPB sodium phosphate buffer     

Identification of salt stress inducible genes that control cell envelope related functions in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

Plant growth promoting rhizobacteria such as Azospirillum brasilense are agronomically important as they are frequently used for crop inoculation. But adverse factors such as increasing soil salinity limit their survival, multiplication and phytostimulatory effect. In order to understand the role of the genes involved in the adaptation of A. brasilense Sp7 to salt stress, a mutant library (6,800 mutants) was constructed after random integration of a mini-Transposon Tn5 derivative containing a promoterless gusA and oriV. The library was screened for salt stress inducible Gus activity on minimal malate agar medium containing NaCl and 5-bromo-4-chloro-3-indolyl-β-d-glucuronide. Salt stress responsiveness of the promoters was estimated by quantifying GusA activity in the presence and absence of NaCl stress using p-nitrophenyl-β-d-glucuronide as a substrate. In 11 mutants showing high levels of gusA expression in the presence of salt-stress, the partial nucleotide sequence of the DNA region flanking the site of Tn5 insertion was determined and analysed using the NCBI-BLAST programs. Similarity searches revealed that 10 out of the 11 genes sequenced showed notable similarity with genes involved in functions related to modulation in the composition of exopolysaccharides, capsular polysaccharides, lipopolysaccharides, peptidoglycan and lipid bilayer of the cell envelope. Induction of cell envelope related genes in response to salt stress and salt sensitive phenotype of several mutants in A. brasilense indicate a prominent role of cell envelope in salt-stress adaptation.     

The Influence of the Synthetic Growth Regulator α-Chloro-(β-(3-chloro-o-tolyl) propionitrile on Some Phytochrome-mediated Processes

In intact Sinapis alba seedlings the synthetic growth regulator α-chloro-β-(3-chloro-o-tolyl)propionitrile (PRB-8) inhibits the phytochrome-mediated anthocyanin synthesis, carotenoid synthesis and L-phenylalanine ammonia-lyase activity. This action was compared with that of other auxins and with that of α-chloro-β-(3-chloro-o-tolyl)propionitrile-homologues. From the results it appeared that PRB-8 and some other o-tolyl derivatives are more inhibitive than other known auxins.     

Halogenated phlorethols and fucophlorethols from the brown alga Cystophora retroflexa.

From an ethyl acetate fraction of the brown alga Cystophora retroflexa several halogenated phlorotannins were isolated. Most of the compounds are derivatives of diphlorethol penta-acetate and triphlorethol-A hepta-acetate. The majority turned out to be chlorinated and/or brominated. Only one iodinated substance, 2-iodophloroglucinol triacetate, was isolated. The structure of this derivative and the following compounds have been characterized previously: 2([D])-bromodiphlorethol penta-acetate, 3([A1])-bromodiphlorethol penta-acetate, 4([D])-bromo-diphlorethol penta-acetate, 4([D])-chlorodiphlorethol penta-acetate, 3([A1])-chlorotriphlorethol-A hepta-acetate, 4([D])-bromotriphlorethol-A hepta-acetate and 4([D])-chlorobisfucopentaphlorethol-A nonadeca-acetate. Ten halogenated phlorethols and two chlorinated fucophlorethols are described for the first time and characterized as their acetates: 2([B])-bromotriphlorethol-A hepta-acetate, 2([D])-bromotriphlorethol-A hepta-acetate, 2([B]), 2([D])-dibromotriphlorethol-A hepta-acetate, 3([A1]), 5([A1])-dichlorotriphlorethol-A hepta-acetate, 3([A1]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 3([A1])-chloro-4([D])-bromotriphlorethol-A hepta-acetate. 2([B]), 4([D])-dichlorotriphlorethol-A hepta-acetate, 2([D]), 3([A1])-dibromotriphlorethol-A hepta-acetate, 3([A1])-bromo-2([D])-chlorotriphlorethol-A hepta-acetate, 2([D])-bromotetraphlorethol-C nona-acetate, 4([D])-chlorofucotriphlorethol-B dodeca-acetate and 4([D])-chlorobisfucotetraphlorethol-A heptadeca-acetate.     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

Olfactory antennal responses to plant volatiles in apterous virginoparae of the vetch aphidMegoura viciae

Electroantennogram (EAG) responses were recorded from apterous virginoparae of the vetch aphidMegoura viciae Buckton (Homoptera, Aphididae) to more than eighty volatile compounds in order to investigate its sensory ability to perceive plant odours. The response profile ofM. viciae reveals a differential sensitivity for the array of plant volatiles tested. The whole group of general green leaf volatiles is very stimulatory. In addition to (E)-2-hexenal, the following compounds of this group elicit large EAG responses: (E)-2-heptenal, 1-octenol-3, hexyl acetate, (Z)-3-hexenyl acetate, hexanol-1, hexanal, 2-heptanone and 3-octanone. Relatively large EAGs are also produced by 4-methoxybenzaldehyde (p-anisaldehyde), hexanonitrile, heptanonitrile, 1,6-hexanedithiol, butyl isothiocyanate, 4-pentenyl isothiocyanate, (−)-(1S)-β-pinene, (+)-(S)-carvone, (−)-(R)-carvone, α-terpineol, linalool and citronellal. The nitriles are the most effective of all plant volatiles tested. Structure-activity relationships occur in various groups of chemicals and members of the green leaf volatiles, benzaldehydes, isothiocyanates and monoterpenes are ranked accordingly. In the group of green leaf volatiles, aliphatic aldehydes are more stimulating than the corresponding alcohols. EAG responses to series of saturated aliphatic alcohols and aldehydes reveal that C₆ and C₇ compounds are the most stimulatory. Dose-response curves show that the rank order of EAG response amplitudes hardly changes at lower dosages. It is concluded thatM. viciae perceives general plant volatiles as well as more-specific components, such as nitriles and isothiocyanates, associated with the odour blends of non-host plant species.