Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.     

Oxidation of Naphthenoaromatic and Methyl-Substituted Aromatic Compounds by Naphthalene 1,2-Dioxygenase

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene.     

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.     

Bacterial oxidation of the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene.

A Beijerinckia sp. and a mutant strain, Beijerinckia sp. strain B8/36, were shown to cooxidize the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene. Both organisms oxidized acenaphthene to the same spectrum of metabolites, which included 1-acenaphthenol, 1-acenaphthenone, 1,2-acenaphthenediol, acenaphthenequinone, and a compound that was tentatively identified as 1,2-dihydroxyacenaphthylene. In contrast, acenaphthylene was oxidized to acenaphthenequinone and the compound tentatively identified as 1,2-dihydroxyacenaphthylene by the wild-type strain of Beijerinckia. Both of these products were also formed when the organism was incubated with synthetic cis-1,2-acenaphthenediol. A metabolite identified as cis-1,2-acenaphthenediol was formed from acenaphthylene by the mutant Beijerinckia sp. strain B8/36. Cell extracts prepared from the wild-type Beijerinckia strain contain a constitutive pyridine nucleotide-dependent dehydrogenase which can oxidize 1-acenaphthenol and 9-fluorenol. The results indicate that although acenaphthene and acenaphthylene are both oxidized to acenaphthenequinone, the pathways leading to the formation of this end product are different.     

Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list

The photomutagenicity of 16 polycyclic aromatic hydrocarbons (PAHs), all on the United States Environmental Protection Agency (US EPA) priority pollutant list, was studied. Concomitant exposing the Salmonella typhimurium bacteria strain TA102 to one of the PAHs and light (1.1 J/cm2 UVA+2.1 J/cm2 visible) without the activation enzyme S9, strong photomutagenic response is observed for anthracene, benz[a]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, and pyrene. Under the same conditions, acenaphthene, acenaphthylene, benzo[k]fluoranthene, chrysene, and fluorene are weakly photomutagenic. Benzo[b]fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenz[a,h]anthracene are not photomutagenic. These results indicate that PAHs can be activated by light and become mutagenic in Salmonella TA102 bacteria. At the same time, the mutagenicity for all the 16 PAHs was examined with the standard mutagenicity test with 10% S9 as the activation system. Benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, acenaphthylene, and fluorene are weakly mutagenic, while the rest of the PAHs are not. In general, the photomutagenicity of PAHs in TA102 does not correlate with their S9-activated mutagenicity in either TA102 or TA98/TA100 since they involve different activation mechanisms.     

Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.     

Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds.

Pseudomonas cepacia F297 grew with fluorene as a sole source of carbon and energy; its growth yield corresponded to an assimilation of about 40% of fluorene carbon. The accumulation of a ring meta-cleavage product during growth and the identification of 1-indanone in growth media and washed-cell suspensions suggest that strain F297 metabolizes fluorene by mechanisms analogous to those of naphthalene degradation. In addition to fluorene, strain F297 utilized for growth a wide variety of polycyclic aromatic compounds (PACs), including naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene, and dibenzothiophene. Fluorene-induced cells of the strain also transformed 2,6-dimethylnaphthalene, biphenyl, dibenzofuran, acenaphthene, and acenaphthylene. The identification of products formed from those substrates (by gas chromatography-mass spectrometry) in washed-cell suspensions indicates that P. cepacia F297 carries out the following reactions: (i) aromatic ring oxidation and cleavage, apparently using the pyruvate released for growth, (ii) methyl group oxidations, (iii) methylenic oxidations, and (iv) S oxidations of aromatic sulfur heterocycles. Strain F297 grew with a creosote-PAC mixture, producing an almost complete removal of all aromatic compounds containing 2 to 3 rings in 14 days, as demonstrated by gas chromatography analysis of the remaining PACs recovered from cultures. The identification of key chemicals confirmed that not only are certain compounds depleted but also the anticipated reaction products are found.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems.

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Degradation of polycyclic aromatic hydrocarbon compounds under various redox conditions in soil-water systems

This study evaluated the microbial degradation of naphthol, naphthalene, and acenaphthene, under aerobic, anaerobic, and denitrification conditions in soil-water systems. Chemical degradation of naphthol and naphthalene in the presence of a manganese oxide was also studied. Naphthol, naphthalene, and acenaphthene were degraded microbially under aerobic conditions from initial aqueous-phase concentrations of 9, 7, and 1 mg/liter to nondetectable levels in 3, 10, and 10 days, respectively. Under anaerobic conditions naphthol degraded to nondetectable levels in 15 days, whereas naphthalene and acenaphthene showed no significant degradation over periods of 50 and 70 days, respectively. Under denitrification conditions naphthol, naphthalene, and acenaphthene were degraded from initial aqueous-phase concentrations of 8, 7, and 0.4 mg/liter to nondetectable levels in 16, 45, and 40 days, respectively. Acclimation periods of approximately 2 days under aerobic conditions and 2 weeks under denitrification conditions were observed for both naphthalene and acenaphthene. Abiotic degradation of naphthalen and naphthol were evaluated by reaction with manganese oxide, a minor soil constituent. In the presence of a manganese oxide, naphthalene showed no abiotic degradation over a period of 9 weeks, whereas the aqueous naphthol concentration decreased from 9 mg/liter to nondetectable levels in 9 days. The results of this study show that low-molecular-weight, unsubstituted, polycyclic aromatic hydrocarbons are amenable to microbial degradation in soil-water systems under denitrification conditions.     

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs

The laccase genes lccα, lccβ, lccγ and lccδ encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccα and Lccβ possessed higher thermal stability, but lower catalytic activity towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccγ and Lccδ. Activities of the laccases were quite different as well. Laccase Lccδ showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccβ. After 72 h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccβ in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs.     

Initial Oxidation Products in the Metabolism of Pyrene, Anthracene, Fluorene, and Dibenzothiophene by the White Rot Fungus Pleurotus ostreatus

The initial metabolites in the degradation of pyrene, anthracene, fluorene, and dibenzothiophene by Pleurotus ostreatus were isolated by high-pressure liquid chromatography and characterized by UV-visible, gas-chromatographic, mass-spectrometric, and (sup1)H nuclear magnetic resonance spectral techniques. The metabolites from pyrene, dibenzothiophene, anthracene, and fluorene amounted to 45, 84, 64, and 96% of the total organic-solvent-extractable metabolites, respectively. Pyrene was metabolized predominantly to pyrene trans-4,5-dihydrodiol. Anthracene was metabolized predominantly to anthracene trans-1,2-dihydrodiol and 9,10-anthraquinone. In contrast, fluorene and dibenzothiophene were oxidized at the aliphatic bridges instead of the aromatic rings. Fluorene was oxidized to 9-fluorenol and 9-fluorenone; dibenzothiophene was oxidized to the sulfoxide and sulfone. Circular dichroism spectroscopy revealed that the major enantiomer of anthracene trans-1,2-dihydrodiol was predominantly in the S,S configuration and the major enantiomer of the pyrene trans-4,5-dihydrodiol was predominantly R,R. These results indicate that the white rot fungus P. ostreatus initially metabolizes polycyclic aromatic hydrocarbons by reactions similar to those previously reported for nonligninolytic fungi. However, P. ostreatus, in contrast to nonligninolytic fungi, can mineralize these polycyclic aromatic hydrocarbons. The identity of the dihydrodiol metabolites implicates a cytochrome P-450 monooxygenase mechanism.     

Treatment of Groundwater Contaminated with PAHs, Gasoline Hydrocarbons, and Methyl tert-butyl Ether in a Laboratory Biomass-Retaining Bioreactor

In this study, we investigated the treatability of co-mingled groundwater contaminated with polycyclic aromatic hydrocarbons (PAHs), gasoline hydrocarbons, and methyl tert-butyl ether (MtBE) using an ex-situ aerobic biotreatment system. The PAHs of interest were naphthalene, methyl-naphthalene, acenaphthene, acenaphthylene, and carbazole. The gasoline hydrocarbons included benzene, toluene, ethyl benzene, and p-xylene (BTEX). Two porous pot reactors were operated for a period of 10 months under the same influent contaminant concentrations. The contaminated groundwater was introduced into the reactors at a flow rate of 4 and 9 l/day, resulting in a hydraulic retention time (HRT) of 32 and 15 h, respectively. In both reactors, high removal efficiencies were achieved for the PAHs (>99%), BTEX and MtBE (>99.7%). All the PAHs of interest and the four BTEX compounds were detected at concentrations less than 1 μg/l throughout the study duration. Effluent MtBE from both reactors was observed at higher levels; nevertheless, its concentration was lower than the 5 μg/l Drinking Water Advisory for MtBE implemented in California.     

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

Bioavailability of polycyclic aromatic hydrocarbons and formation of humic acid-like residues during bacterial PAH degradation

 The degradation of single polycyclic aromatic hydrocarbons (PAHs: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) and a mixture of all seven PAHs by a bacterial culture enriched from contaminated soil resulted in the formation of a dark-coloured residual fraction of dissolved (DOM) and particulate organic matter (POM). This fraction was highly resistant to bacterial degradation. Analysis of the DOM revealed a molecular-size-distribution similar to that of natural humic acids. A complete degradation of PAHs was apparently prevented by an irreversible incorporation of about 10% of the carbon from single PAHs or 20% of the carbon from the mixture of seven PAHs into the DOM- and POM- fraction. Some metabolites excreted during bacterial PAH-degradation were identified as known precursors for humification. Received: 1 June 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Conversion of polycyclic aromatic hydrocarbons by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. VKM B-2434

A versatile bacterial strain able to convert polycyclic aromatic hydrocarbons (PAHs) was isolated, and a conversion by the isolate of both individual substances and PAH mixtures was investigated. The strain belonged to the Sphingomonas genus as determined on the basis of 16S rRNA analysis and was designated as VKM B-2434. The strain used naphthalene, acenaphthene, phenanthrene, anthracene and fluoranthene as a sole source of carbon and energy, and cometabolically oxidized fluorene, pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene. Acenaphthene and fluoranthene were degraded by the strain via naphthalene-1,8-dicarboxylic acid and 3-hydroxyphthalic acid. Conversion of most other PAHs was confined to the cleavage of only one aromatic ring. The major oxidation products of naphthalene, phenanthrene, anthracene, chrysene, and benzo[a]pyrene were identified as salicylic acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid, o-hydroxyphenanthroic acid and o-hydroxypyrenoic acid, respectively. Fluorene and pyrene were oxidized mainly to hydroxyfluorenone and dihydroxydihydropyrene, respectively. Oxidation of phenanthrene and anthracene to the corresponding hydroxynaphthoic acids occurred quantitatively. The strain converted phenanthrene, anthracene, fluoranthene and carbazole of coal-tar-pitch extract.     

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.     

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.     

Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.