Mineralization of biphenyl and PCBs by the white rot fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium is unique in its ability to totally degrade a wide variety of recalcitrant pollutants. We have investigated the degradation of biphenyl and two model chlorinated biphenyls, 2,2',4,4'-tetrachlorobiphenyl and 2-chlorobiphenyl by suspended cultures of P. chrysosporium grown under conditions that maximize the synthesis of lignin-oxidizing enzymes. Radiolabeled biphenyl and 2'-chlorobiphenyl added to cultures at concentrations in the range 260 nM to 8.8 muM were degraded extensively to CO(2) within 30 days. In addition, from 40% to 60% of the recovered radioactivity was found in water-soluble compounds. A correlation between the rate of degradation and the synthesis of ligninases or Mn-dependent peroxidases could not be observed, indicating that yet unknown enzymatic system may be resonsible for the initial oxidation of PCBs. The more heavily chlorinated PCB congener, 2,2',4,4'-tetrachlorobiphenyl was converted to CO(2) less readily; approximately 9% and 0.9% mineralization was observed in cultures incubated with 40 nM and 5.3 muM, respectively. Overall, our results indicate that P. chrysosporium is a promising organism for the treatment of wastes contaminatd with lightly and moderately chlorinated PCBs. (c) 1992 John Wiley & Sons, Inc.     

Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

Abstract Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana . Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium . Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium , despite differences between brown and white rot modes of decay.     

Functional diversity of cytochrome P450s of the white-rot fungus Phanerochaete chrysosporium

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda⁺ isolates) hybridized to PdaT9, while only one Pda^– isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda^– phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

Respiratory pathways and oxygen toxicity in Phanerochaete chrysosporium

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Degradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium grown in ammonium lignosulphonate media

Removal and degradation of pentachlorophenol (PCP) by Phanerochaete chrysosporium in static flask cultures was studied using ammonium lignosulphonates (LS), a waste product of the papermill industry, as a carbon and nitrogen source. After 3 days, cultures of P. chrysosporium grown in either a 2% LS (nitrogen-sufficient) medium or a 0.23% LS and 2% glucose (nitrogen-deficient) medium removed 72 to 75% of PCP, slightly less than the 95% removal seen using nitrogen-deficient glucose and ammonia medium. PCP dehalogenation occurred despite the fact that extracellular enzyme (LiP) activity, measured by a veratryl alcohol oxidation assay and by PCP disappearance in cell-free extracts, was inhibited by LS. This inactivation of LiP likely contributed to the lower percent of PCP dehalogenation observed using the LS media. In order to better understand the relationship between PCP disappearance and dehalogenation, we measured the fate of the chlorine in PCP. After 13 days, only 1.8% of the initial PCP added was recoverable as PCP. The remainder of the PCP was either mineralized or transformed to breakdown intermediates collectively identified as organic halides. The largest fraction of the original chlorine (58%) was recovered as organic (non-PCP) halide, most of which (73%) was associated with the cell mass. Of the remaining chlorine, 40% was released as chloride ion, indicating a level of dehalogenation in agreement with previously reported values.     

Role of cytochrome P-450 in ochratoxin a-stimulated lipid peroxidation

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe³⁺, ethylenediaminetetra-acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe³⁺. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

Some aspects of the role of cytochrome P-450 isozymes in the n-oxidative transformation of secondary and tertiary amine compounds

Indirect evidence of the participation of cytochrome P-450 (P-450) in the microsomal N-oxygenation of secondary and tertiary nitrogen functions is presented by studies employing diagnostic modifiers of the hemoprotein system as well as antibodies directed toward the diverse P-450 isoforms and NADPH-cytochrome P-450 reductase. Experiments with recombinant hemoproteins or P-450 isozymes directly purified from the tissues of various animal species support the results obtained by the inhibitor assays. Although the intermediacy of aminium radicals is thought to be restrictive to P-450-catalyzed N-oxygenation of secondary and tertiary amine groups bearing accessible hydrogens on the α-carbon, numerous exceptions to this rule are documented. It is proposed that aminium radicals partition between oxygen rebound and α-hydrogen abstraction to yield a finite level of N-oxygenated product in all P-450-mediated amine oxidations, the partition ratio depending on the amine structure and particular P-450 isozyme operative. In some instances, N-oxygenation appears to proceed by peroxidatic mechanisms. The relative contribution of P-450 to the N-oxygenation of secondary and tertiary amines in crude preparations or live animals, where competition with the flavin-containing monooxygenase (FMO) occurs, seems to be a function of the relative amounts and catalytic capacities of the two enzyme systems. Both parameters are species and tissue dependent. Accordingly, the extent to which P-450 contributes to total N-oxidative turnover of the amine substrates varies from minor to major. © 1996 John Wiley & Sons, Inc.     

Development of bacterial cytochrome P-450(cam) (cytochrome m) production

Cytochrome P-450(cam) monooxygenase is an important bacterial redox enzyme system with potential commercial value for detoxifying trace hydrocarbon contaminants, catalyzing regiospecific hydroxylations, and amperometric biosensing. The present study was undertaken to increase productivity of this enzyme, which is induced in its host, pseudomonas putida PpG 786, by D(+)-camphor. Culture processes were studied in batch, fed-batch, and continuous modes to evaluate metabolic behavior and develop constitutive equations for specific rate of growth (mu), camphor utilization (q(p)). Fed-batch culture was characterized by an extended linear growth phase which is often encountered in hydrocarbon fermentations. Inhibition by the camphor solvent, dimethylformamide, was assessed. Production of the terminal protein of the p-450(cam) enzyme system, cytochrome m, was shown to depend on growth medium iron content in fed-batch culture and was increased by 130% over previously protocols by eliminating iron deficiency. A continuous process that enables greater production rates was developed by using oxygen enrichment while simultaneously reducing gas throughput. Camphor and oxygen requirements were determined for fedbatch and continuous growth. (c) 1993 John Wiley & Sons, Inc.     

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.