Enzymatic degradation of aspergillic acid by Trichoderma koningii M102

Aspergillic acid-degrading enzyme was extracted from the cells of T. koningii M102 grown on a medium containing aspergillic acid. The enzyme was partially purified about 36-fold by ammonium sulfate precipitation and chromatographies with DEAE-Toyopearl 650 M and Toyopearl HW 60 F. The degradation product of aspergillic acid was isolated from chloroform extract of the enzyme reaction mixture, and identified as 2-hydroxyimino-3-methyl-1-pentanal by comparison of the mass spectrum with that of a chemically-synthesized authentic sample.     

Microbial degradation of aspergillic acid by Trichoderma koningii M 102

A microorganism, strain M 102, capable of degrading aspergillic acid (AA), was first isolated from a soil sample in a drainage ditch and was identified as Trichoderma koningii Oudemans. This fungus degraded AA, but not hydroxyaspergillic acid (HAA) or deoxyaspergillic acid (DAA). The AA-degrading ability of M 102 was induced by incubation with AA but not with HAA or DAA. AA-degradation activity was found in a crude enzyme prepared from the mycelia induced by AA; this AA degradation reaction required NAD(P)H and oxygen.     

Purification and Characterization of an Enzyme That Catalyzes Ring Cleavage of Aspergillic Acid,from Tvichoderma koningii ATCC 76666

The aspergillic acid degrading enzyme (ADE) that catalyzes the cleavage of the pyrazine ring in aspergillic acid (AA, l-hydroxy-3-isobutyl-6-sec-butyl-2-pyrazinone) was purified to electrophoretic homogeneity from extracts of Trichoderma koningii ATCC 76666. ADE was a homodimeric protein with a molecular mass of 112kDa, contained lmol of FAD per mol of subunit, and required NAD(P)H and molecular oxygen for its activity. ADE had an isoelectric point of around 5.3, and an optimum pH of 7.0–8.0. p-Chloromercuribenzoate and HgCl₂ completely inhibited ADE activity, while metal chelating reagents, α, α′-dipyridyl and o-phenanthroline, were not inhibitors. The substrate specificity among AA-related compounds was that hydroxyaspergillic acid was a poor substrate (16% of the activity for AA) and deoxyaspergillic acid did not serve as a substrate.     

Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C₁ unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C₁₁H₁₆O₄ (C₁₁H₁₆O₄-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by β-oxidation of one of the side chains of the C₁₁H₁₆O₄-diacid. Stable isotope-labeling growth experiments with either ¹³C-labeled naphthalene, per-deuterated naphthalene-d₈, or a ¹³C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

Optimization of formation and regeneration of protoplasts from biocontrol agents ofTrichoderma species

The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×10⁸/ml fromT. koningii and 6×10⁷/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells.     

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-¹⁴C]cresol was recovered predominantly as [¹⁴C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.     

Koninginins L and M,two polyketides from Trichoderma koningii 8662

Two new fungal metabolites, named koninginins L (1) and M (2), together with three known koninginins A (3), E (4), and trichodermaketone C (5), were isolated from solid fermentation products of Trichoderma koningii 8662. Koninginins L (1) and M (2) were elucidated by extensive spectroscopic methods, including 1D and 2D NMR, HR-EI-MS experiments, and the absolute configuration of compound 1 was confirmed by single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation.     

Isolation,characterisation of major secondary metabolites of the Himalayan Trichoderma koningii and their antifungal activity

Ethyl acetate extracts of two strains of Trichoderma koningii were evaluated for antifungal activity against soilborne pathogenic fungi, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum by poisoned food technique. Secondary metabolites, namely δ-decanolactone, 6-pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one were isolated from T. koningii (T-8) extract while palmitic acid, 6-pentyl-α-pyranone and stigmasterol were isolated from T. koningii (T-11) extract. Secondary metabolites were identified by ¹H NMR, ¹³C NMR and mass spectroscopic methods. These metabolites were evaluated for antifungal activity against the test pathogens 6-Pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one exhibited excellent antifungal activity against S. rolfsii. The antifungal activity though slightly less was comparable to that of hexaconazole, a commercial fungicide.     

Biosynthesis of a Cyclic Tautomer1of (3-Methylmaleyl)acetone from 4-Hydroxy-3,5-dimethylbenzoate byPseudomonassp. HH35 but Not byRhodococcus rhodochrousN75

Here we report that the bacterial catabolism of 4-hydroxy-3,5-dimethylbenzoic acid 1 takes a different course inRhodococcus rhodochrousN75 andPseudomonassp. strain HH35. The former organism accumulates a degradation metabolite of the acid which we isolated and identified as 2,6-dimethylhydroquinone 2. The latter bacterial strain converts the acid and the hydroquinone into a dead-end metabolite. This novel compound was characterised unequivocally by mass spectrometry and¹H and¹³C NMR and UV spectroscopy as 4-acetonyl-4-hydroxy-2-methylbut-2-en-1,4-olide 4, a cyclic tautomer of (3-methylmaleyl)acetone, which exists as the enol carboxylate form 3 in aqueous solution.     

midD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K _m, V _max and K _cat of MidD were 1.27 × 10^?4 mol, 4.96 × 10^?5 mol s^?1 mg^?1, and 2,256.05 s^?1, respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5′-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including l-tyrosine, l-tryptophan and l-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C–N lyase.     

High-performance liquid chromatographic determination and identification of acyl migration and photodegradation products of furosemide 1-O-acyl glucuronide

Stability of furosemide glucuronide, the major metabolite of furosemide, was studied in order to accurately assess the glucuronidation of furosemide. Furosemide glucuronide was purified by high-performance liquid chromatography, and the mass spectrum of furosemide glucuronide showed the molecular ion peaks [M−H]⁻ at 505 and 507 (m/z). Furosemide glucuronide was photodegraded to the compound, which was shown more hydrophilic than furosemide glucuronide by high-performance liquid chromatography assay. The photodegradation product of furosemide glucuronide was hydrolyzed to one of the photodegradation products of furosemide by β-glucuronidase, indicating that the photodegradation product of furosemide glucuronide possessed a glucuronic acid moiety. Furthermore, the mass spectrum of the photodegradation product of furosemide glucuronide exhibited molecular ion peaks [M−H]⁻ at 487 and [M−2H+2Na]⁻ at 509, indicating the chlorine displacement of furosemide glucuronide by a hydroxyl group. Furosemide glucuronide was unstable in an aqueous solution (pH=7.4), and presumed acyl migration isomers of furosemide glucuronide (furosemide glucuronide-isomers) were detected by high-performance liquid chromatography equipped with photodiode array UV detector. The UV spectra of seven furosemide glucuronide-isomers were closely similar to that of furosemide glucuronide but not furosemide. Exposing a mixture of furosemide glucuronide and furosemide glucuronide-isomers to light resulted in the production of new compounds. UV spectra of photodegradation products of furosemide glucuronide-isomers were closely similar to those of photodegradation product of furosemide glucuronide. These results suggested that furosemide glucuronide-isomers were also photodegraded, resulting in the displacement of chlorine by a hydroxyl group as in furosemide glucuronide.     

Biological and fungicidal antagonism of Sclerotium cepivorum for controlling onion white rot disease

The action of some microbial isolates and Topsin-M against the most pathogenic isolate (Sc₂) of Sclerotium cepivorum causing onion white rot was tested. Bacillus subtilis B₄, B. subtilis B₅, Trichoderma koningii and Trichoderma harzianum were the most antagonistic isolates of the causal fungus. Mycelia of sclerotial germination of S. cepivorum (Sc₂) were completely inhibited in vitro by application of 2.0 g ?L^?1 Topsin-M. In pots, disease incidence was decreased to 8.33 % by the use of Topsin-M followed by T. koningii (29.17 %) compared with 95.83 % for the control, i.e., a remarkable reduction in severity was obtained. Under field conditions, disease incidence was decreased to 2.78 % by Topsin-M and to 11.11 % by T. harzianum. Both agents caused a sharp reduction in disease severity, reaching 1.39 % and 9.72 %, respectively, with 11.80 % being achieved by T. koningii and B. subtilis B₅. A close link between the biological action and enhancement of the enzyme activities of polyphenol oxidase and peroxidase with ability of onion to resist S. cepivorum was found, indicating induction of systemic acquired resistance. Accordingly, chlorophyll, root and foliage lengths, foliar, bulb dry matter and bulb productivity were also enhanced upon application of this biological control strategy.     

THYMIDINE DEGRADATION PRODUCTS IN PLANT TISSUES LABELED WITH TRITIATED THYMIDINE

A study of the metabolic pathways of H³-thymidine utilization in buds of Lilium longiflorum and root tips of Vicia faba was undertaken in order to obtain information that might explain the binding of H³ from H³-thymidine in the cytoplasm of these plants. H³-thymidine was administered for various periods of time, the tissues were fixed and processed in the manner routinely used in preparation for sectioning and autoradiography, and the radioactivity removed in this way from the tissues was determined. It was found that the ethanol/acetic acid fixative contained the major portion of the radioactivity. Analysis of this extract by paper chromatography showed that the radioactivity was distributed among various degradation products of thymidine, principally β-ureidoisobutyric acid and β-aminoisobutyric acid. Time course experiments with Vicia showed that these degradation products rapidly appeared in the tissue during incubation with H³-thymidine, while H³-thymine appeared in the incubation medium. Preliminary studies indicated that Vicia root tips incubated with H³-dihydrothymine for 24 hours would bind a small amount of H³ non-specifically in the cells. It seems unlikely that utilization of degradation products of H³-thymidine is sufficient to explain labeling which is concentrated in the cytoplasm.     

Cytogenetic effect of radiation products of cytosine: Parabanic and oxaluric acids

The two final products of radiolytic degradation of cytosinei.e. parabanic and oxaluric acids were investigated as regards their cytogenetic effects. Broad bean (Vicia faba L.) root meristem was used as experimental material. While the oxaluric acid was not able to induce chromosomal aberrations to a greater extent, the parabanic acid acts as a clastogenic compound. When applied on a resting broad bean root meristem at concentration from 10^-3 to 10^-2 M it induces chromosome and chromatid aberrations, especially breakages. Their frequency reaches 9–12% at the highest tested concentration. The same concentrations of the parabanic acid increases mitotic index of the first cell generation of primary root.     

康宁木霉对龟裂链霉菌MY02诱导活性及其诱导组分的初步分析

利用真菌作为诱导菌株评价其对龟裂链霉菌(Streptomyces rimosus) MY02的活性代谢产物的影响,并对其诱导条件进行优化。当以康宁木霉(Trichoderma koningii)和香菇真菌(Lentinus edodes)作为诱导菌株,黄瓜枯萎病菌(Fusarium oxysporum f sp.cucumarinum)作为指示菌,以菌株MY02作为被诱导菌株时,康宁木霉和香菇真菌对菌株MY02的抗真菌活性均具有明显的正向诱导作用,菌株MY02的抗真菌活性均比对照明显增强。以康宁木霉作为诱导菌株时,其最佳诱导条件为接种量2%,添加时间为发酵36 h。通过初步分析康宁木霉的诱导组分为其发酵产生的多糖类物质。     

Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter

We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.     

Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11

CYP102A1, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed drug-metabolizing mutant CYP102A1 M11 by a combination of site-directed and random mutagenesis. CYP102A1 M11 contains eight mutations, when compared with wild-type CYP102A1, and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug-metabolizing mutant CYP102A1 M11 was mutated to all possible natural amino acids to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants, extremely fast rates of NADPH oxidation were observed (up to 6,000 min⁻¹). However, the coupling efficiencies were extremely low: even for the substrates showing the highest rates of O-dealkylation, coupling efficiencies were lower than 1%. With testosterone as the substrate, all mutants were able to produce three hydroxytestosterone metabolites, although with different activities and with remarkably different product ratios. The results show that the nature of the amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant CYP102A1 M11. Because of the wide substrate selectivity of CYP102A1 M11 when compared with wild-type CYP102A1, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.     

Degradation of the novel herbicide ZJ0273 by Amycolatopsis sp. M3-1 isolated from soil

ZJ0273, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate, is a novel and broad-spectrum herbicide. In this study, 15 bacteria capable of utilizing ZJ0273 as the sole carbon source were isolated from soil. One of the isolates belonged to the family Amycolatopsis and was designated to Amycolatopsis sp. M3-1; at 30°C and pH 7.0, degradation rate of ZJ0273 could reach at 59.3% and 68.5% in 25 days and 60 days, respectively. Furthermore, six metabolites (M1–M6) during the degradation of ZJ0273 by Amycolatopsis sp. M3-1 were identified by a combination with multi-position ¹⁴C-labeled compounds (B-ZJ0273 and C-ZJ0273), chromatography, liquid scintillation spectrometer, and LC–MS, a novel pathway of ZJ0273 degradation by Amycolatopsis sp. M3-1 was proposed based on the identified metabolites and their biodegradation courses. ZJ0273 was initially hydrolyzed into M1 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoic acid), then further oxidized into M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid). M1 also could undergo a carbonylation into M2 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzamido) benzoic acid), and then its C–N and C–O bonds were cleaved to yield M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid) and M4 (4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5 (2-(4-hydroxy, 6-methoxypyrimidin-2-yloxy) benzoic acid) and M6 (2, 4-dihydroxy-pyrimidine) were found. M5 was formed through de-methyl of M3 and then hydrolyzed into M6.     

Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity

Background

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ⁹-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2''s high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).

Methods/Principal Findings

JWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [³H]CP-55,940, and then for CB1R intrinsic efficacy using an [³⁵S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting K_i values that were lower than or equivalent to Δ⁹-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ⁹-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ⁹-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.

Conclusions/Significance

Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ⁹-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.     

New indanone compounds from Onychium japonicum

A new indanone glucoside pteroside M has been isolated from fronds of Onychium japonicumPteridaceae. The structure of its aglycone pterosin M has been established by ¹³C NMR, PMR spectra and degradation with nitric acid.