Fungal secondary metabolism - from biochemistry to genomics

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.     

1-(Benzofuran-2-yl)-2-(3,3,3-trifluoropropyl)aminopentane HCl, 3-F-BPAP,antagonizes the enhancer effect of (-)-BPAP in the shuttle box and leaves the effect of (-)-deprenyl unchanged

The subcutaneous administration of 1 mg/kg tetrabenazine, once daily for 5 days, which depletes the catecholamine stores in the brain, significantly inhibits in rats the acquisition of a two-way conditioned avoidance reflex in the shuttle box. Enhancer substances, the tryptamine-derived selective and highly potent enhancer, R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane HCl [(-)-BPAP] (0.05-10 mg/kg), the beta-phenylethylamine (PEA)-derived enhancer, (-)-deprenyl (1-5 mg/kg) and the (-)-deprenyl analogue, free of MAO-B inhibitory potency, (-)-1-phenyl-2-propylaminopentane HCl [(-)-PPAP], (1-5 mg/kg), antagonize in a dose-dependent manner the inhibition of learning caused by tetrabenazine. 1-(Benzofuran 2 yl)-2-(3,3,3-trifluoropropyl)aminopentane HCl [3 F BPAP], a newly synthetized analogue of (-)-BPAP with low specific activity, significantly antagonized the enhancer effect of (-)-BPAP but left the effect of (-)-deprenyl and (-)-PPAP unchanged. This is the first proof for a difference in the mechanism of action between a PEA-derived enhancer substance and its tryptamine-derived peer.     

L-dopa metabolism in genetically hypertensive mice: effect of pargyline

This study on the role of the sympathetic nervous system in the development of hypertension involves the measurement of dopamine and norepinephrine accumulation in various tissues of the hypertensive and random-bred normotensive strains of mice at basal levels, and following a pargyline-L-dopa treatment. Under such a treatment, designed to suppress the homeostatic action of monoamine oxidase and to better expose the relationship between dopamine and norepinephrine, the brain and heart of the hypertensive mice accumulated more dopamine than the normotensive mice. There was a significantly lower norepinephrine accumulation in the heart of the hypertensive mice in spite of comparable dopamine-beta-hydroxylase activity in this tissue between the two strains of mice. Under the pargyline-L-dopa treatment, the brain and heart of the older mice in both hypertensive and normotensive strains accumulated significantly (p less than 0.05) more dopamine than those of their younger counterparts, while their norepinephrine accumulation remained unchanged. The results demonstrated different patterns of response of dopamine and norepinephrine in the development of hypertension.     

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).     

Fungal metabolism of biphenyl.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.     

Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

A pharmacological analysis elucidating why,in contrast to (-)-deprenyl (selegiline), alpha-tocopherol was ineffective in the DATATOP study

The Parkinson Study Group who conducted the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) trial designed their study in the belief that the MAO inhibitor (-)-deprenyl (selegiline), the antioxidant alpha-tocopherol, and the combination of the two compounds will slow the clinical progression of the disease to the extent that MAO activity and the formation of oxygen radicals contribute to the pathogenesis of nigral degeneration. In fact, (-)-deprenyl only delayed the onset of disability associated with early, otherwise untreated Parkinson's disease, however, in contrast to the expectation of the authors, alpha-tocopherol proved to be ineffective in the DATATOP study. Enhancer substances, (-)-deprenyl, (-)-1-phenyl-2-propylaminopentane [(-)-PPAP] the (-)-deprenyl analogue free of MAO inhibitory potency, and R-(-)1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] the presently known most potent enhancer substance, are peculiar stimulants. They enhance the impulse propagation mediated release of the catecholamines in the brain. Due to their enhancer effect, the amount of catecholamines released from selected discrete brain areas (striatum, substantia nigra, tuberculum olfactorium, locus coeruleus) is significantly higher in rats treated with an enhancer substance than in saline treated rats. We compared the effect of (-)-deprenyl 0.025 and 0.25 mg/kg, (-)-PPAP 0.1 mg/kg, (-)-BPAP 0.0001 mg/kg, and alpha-tocopherol 25 and 50 mg/kg, in this test. The doses of (-)-deprenyl and alpha-tocopherol were selected to be in compliance with the dose given in the DATATOP study. Compared to saline treated rats, the enhancer substances significantly increased the amount of dopamine released from the striatum, substantia nigra and tuberculum olfactorium and the amount of norepinephrine released from the locus coeruleus; alpha-tocopherol was ineffective. The results indicate that alpha-tocopherol was ineffective, because, unlike (-)-deprenyl it dose not enhance the activity of the nigrostriatal dopaminergic neurons.     

Enantioselective metabolism of (R)- and (S)-4-hydroxy-2-nonenal in rat

4-Hydroxy-2-nonenal (HNE) is an endogenous product of lipid peroxidation, which is believed to play a biological role in the pathogenesis of various diseases. HNE is formed as a racemic mixture of (R)- and (S)- enantiomers. These enantiomers differ in their biological properties. The aim of this study was to investigate separately the in vivo metabolism of the two HNE enantiomers in male rats after intravenous administration of the corresponding radiolabeled compounds and to compare the results with those obtained with the racemic mixture. Although the difference in the excretion rates was not statistically significant, the HPLC profiles of urinary metabolites showed qualitative and quantitative differences between the two enantiomers. The level of 3-mercapturic acid-1,4-dihydroxynonane, which is considered as the major urinary metabolite of HNE, was significantly lower in the case of (S)-HNE injected rats. In vitro studies using rat liver cytosolic incubations and HNE-glutathione conjugate as substrate were performed to clarify the intermediate pathways involved in their metabolism. Large differences were obtained in the reduction and retro-Michael conversion steps of the metabolism between the conjugates originating from the two enantiomers.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Fungal Extracellular RNases (Review)

Studies of certain fungal extracellular RNases mainly isolated from representatives of the genera Aspergillus and Penicillium are reviewed. The isolation of these enzymes in highly purified states in the 1970s and 1980s strongly stimulated further studies of their structure, functions, and mechanisms of action. This also promoted the use of RNases as catalysts in oligoribonucleotide syntheses and as objects of comparative and evolutionary biochemical and other studies. Results of determinations of the primary, secondary, and spatial structures of guanyl-specific fungal RNases are reviewed. These studies revealed considerable homology within the subfamilies of fungal, bacterial, and actinomycete RNases. Characteristics of the nonspecific RNase Pb₂ are considered.     

Fungal extracellular ribonucleases (Review)

Results of studies of certain fungal extracellular ribonucleases mainly isolated from representatives of the genera Aspergillus and Penicillium are summarized. The isolation of these enzymes in highly purified states in the 1970-1980s strongly stimulated further studies of their structure, functions, and mechanisms of action. This also promoted the use of ribonucleases as catalysts in oligoribonucleotide syntheses and as objects of comparative and evolutionary biochemistry and other research works. Results of studies of the primary, secondary, and spatial structures of guanyl-specific fungal ribonucleases are reviewed. These studies revealed a high homology within the subfamilies of fungal, bacterial, and actinomycete RNases. Characteristics of the nonspecific Pb2 RNase are considered.     

In vitro oxidative metabolism study of (-)-rhazinilam

Metabolism studies were conducted in order to investigate the reasons for the in vivo lack of activity of (-)-rhazinilam 1, an original poison of the mitotic spindle. Bioconversion by Beauveria bassiana strains, rat and human liver microsomes allowed the identification of metabolites 2, 3, and 4 oxidized in positions 3 and 5 of rhazinilam. Further experiments indicated that CYP2B6 was the main CYP responsible for the oxidation of 1 by human liver microsomes. All isolated metabolites were markedly less active than rhazinilam in vitro, which might explain its in vivo inactivity.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate.

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.