Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Cometabolic Degradation of Dibenzofuran by Biphenyl-Cultivated Ralstonia sp. Strain SBUG 290

Cells of the gram-negative bacterium Ralstonia sp. strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated. Meta cleavage of the 1,2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid, which was degraded completely via salicylic acid. The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by Ralstonia sp. strain SBUG 290.     

3-(2-hydroxyphenyl)catechol as substrate for proximal meta ring cleavage in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361.

Brevibacterium sp. strain DPO 1361 oxygenates dibenzofuran in the unusual angular position. The 3-(2-hydroxyphenyl)catechol thus generated is subject to meta ring cleavage in the proximal position, yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, which is hydrolyzed to 2-oxo-4-pentenoate and salicylate by 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase. The proximal mode of ring cleavage is definitely established by isolation and unequivocal structural characterization of a cyclization product of 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, i.e., 3-(chroman-4-on-2-yl)pyruvate.     

Oxidation of Indole-3-acetic Acid and Oxindole-3-acetic Acid to 2,3-Dihydro-7-hydroxy-2-oxo-1H Indole-3-acetic Acid-7′-O-β-d-Glucopyranoside in Zea mays Seedlings

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7′-O-β-d-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid → Oxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid-glucoside.     

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.     

Biotransformation of biphenyl by the filamentous fungus <Emphasis Type="Italic">Talaromyces helicus</Emphasis>

The filamentous fungusTalaromyces helicus , isolated from oil-contaminated sludge, oxidizes biphenyl via 4-hydroxybiphenyl to the dihydroxylated derivatives 4,4-dihydroxybiphenyl and 3,4-dihydroxybiphenyl, which, to a certain extent, are converted to glycosyl conjugates. The sugar moiety of the conjugate formed from 4,4-dihydroxybiphenyl was identified as glucose. Further metabolites: 2-hydroxybiphenyl, 2,5-dihydroxylated biphenyl, and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces. From these results the main pathway for biotransformation of biphenyl in T. helicus could be proposed to be the excretion of dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).     

Oxidation and ring cleavage of dibenzofuran by the filamentous fungus <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>

The ability of the imperfect soil fungus Paecilomyces lilacinus to transform the environmental pollutant dibenzofuran was investigated. Transformation of dibenzofuran and related derivatives lead to 14 products, which were identified by UV spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Biotransformation was initiated by two separate hydroxylation steps, leading to the accumulation of 4-monohydroxylated and 4-dihydroxylateddibenzofurans. Hydroxylation at both aromatic rings produced 2,7-dihydroxydibenzofuran, 3,7-dihydroxydibenzofuran, and 2,8-dihydroxydibenzofuran. Further oxidation yields ring cleavage of dibenzofuran, which has not been described before for filamentous fungi. The ring fission products were identified as benzo[b]furo[3,2-d]-2-pyrone-6-carboxylic acid and [2-(1-carboxy-methylidene)-benzofuran-3-ylidene]-hydroxy-acetic acid and its derivatives hydroxylated at carbon 7 and 8 at the non-cleaved ring. Other metabolites were riboside-conjugates of 2-hydroxydibenzofuran and 3-hydroxydibenzofuran. The results showed that P. lilacinus transforms the hydrophobic compound dibenzofuran by phase I/phase II reactions to produce hydroxylated products and excretable sugar conjugates.     

Phenolic metabolites of the bird's nest fungus Nidula niveo-tomentosa

The metabolites of the bird's nest fungus Nidula niveo-tomentosa have been examined. Niduloic acid (3-hydroxy-5-(p-hydroxyphenol) pentanoic acid) is a new natural product. 4-(p-Hydroxyphenyl)-2-butanone, 4-(p-hydroxyphenyl)-2-butanol, trans-4-p-hydroxyphenylbut-3-en-2-one, 4-(3′,4′-dihydroxyphenyl)-2-butanol, 4-(3′,4′-dihydroxyphenyl)-2-butanone, zingerone 3-(p-hydroxyphenyl)-1,2-propanediol are also metabolites of this fungus. An interesting reductive cyclization of zingerone, effected by iodotrimethylsilane, is reported.     

Isolation of Terrabacter sp. Strain DDE-1, Which Metabolizes 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene when Induced with Biphenyl

Terrabacter sp. strain DDE-1, able to metabolize 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) in pure culture when induced with biphenyl, was enriched from a 1-1-1-trichloro-2,2-bis(4-chlorophenyl)ethane residue-contaminated agricultural soil. Gas chromatography-mass spectrometry analysis of culture extracts revealed a number of DDE catabolites, including 2-(4′-chlorophenyl)-3,3-dichloropropenoic acid, 2-(4′-chlorophenyl)-2-hydroxy acetic acid, 2-(4′-chlorophenyl) acetic acid, and 4-chlorobenzoic acid.     

Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes

The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.     

Ascocorynin,a terphenylquinone from Ascocoryne Sarcoides

Ascocorynin, 2,5-dihydroxy-3-(p-hydroxyphenyl)-6-phenyl-1,4-benzoquinone, has been isolated from the fungus Ascocoryne sarcoides. The biogeneti     

The inhibition of gibberellin plant hormone biosynthesis by ent-6-oxo-5β(H)-7-norgibberell-16-enes

Ent-19-hydroxy-6-oxo-5β(H)-7-norgibberell-16-ene and the corresponding 19-carboxylic acid are shown to be inhibitors of gibberellin biosynthesis in the fungus, Gibberella fujikuroi, at stages involved in the oxidative modification of ring B of the kaurenoid precursors.     

Microbial Reduction of 1,3-Dioxo-2-Methyl-2-(3′-Oxo-6′-Carbomethoxyhexyl)-Cyclopentane to Form 1β-Hydroxy-3-Oxo-2β-Methyl-2α-(3′-Oxo-6′-Carbomethoxy-hexyl)-Cyclopentane, an Intermediate for Steroid Total Synthesis

The rate and extent of stereoselective reduction of 1,3-dioxo-2-methyl-2-(3′-oxo-6′-carbomethoxyhexyl)-cyclopentane to form the 1β-hydroxy-2β-methyl isomer by cultures of Schizosaccharomyces pombe ATCC 2476 was dramatically increased by addition to the fermentation of certain α,β-unsaturated ketones and allyl alcohol.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Transformation of the Ionic X-Ray Contrast Agent Diatrizoate and Related Triiodinated Benzoates by Trametes versicolor

Iodinated X-ray contrast agents are considered to be nondegradable by microorganisms. The decomposition of the ionic X-ray contrast agents Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoic acid) and Iodipamide (3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid) and related triiodinated benzoates (Acetrizoate [3-acetylamino-2,4,6-triiodobenzoic acid] and Aminotrizoate [3-amino-2,4,6-triiodobenzoic acid]) by Trametes versicolor has been investigated. The fungus was able to transform all tested triiodinated benzoates cometabolically. During transformation of these compounds, iodide was released, but deiodination was not complete. T. versicolor liberated traces of ¹⁴CO₂ from uniformly ring-¹⁴C-labeled Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoate). Various extracellular metabolites were detected during transformation of the different substances. In the transformation of Diatrizoate, the three main metabolites were identified as 3,5-di(acetamido)-2,6-diiodobenzoic acid, 3,5-di(acetamido)-2,4-diiodobenzoic acid, and 3,5-di(acetamido)-2-iodobenzoic acid, suggesting reductive deiodinations in steps as initial transformation steps.     

Aerobic Biodegradation of 2,2′-Dithiodibenzoic Acid Produced from Dibenzothiophene Metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2′-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2′-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2′-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B₁₂, and it degraded 2,2′-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2′-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2′-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2′-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein–DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5′-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a ‘zero-length’ photoreactive group (4-thio-2′-deoxyuridine-5′-monophosphate) embedded in the 3′ end of the DNA primer, or a group connected to the 3′ end by a lengthy linker (5-{N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxyuridine-5′-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C·p32D·p14) was capable of correctly recognizing the primer– template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D·p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.     

The MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for Colorimetric Determination of Fungal Cell Densities

The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting.