Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains.

Four bacterial strains that use picric acid as their sole carbon and energy source were isolated. Mineralization of 14C-UL-picric acid showed that up to 65% of the radioactivity was released as 14CO2. HPLC and UV/Vis spectral analyses indicated complete degradation of picric acid by these organisms. HPLC and LC/MS analyses showed transient formation of 2,4-dinitrophenol during picric acid degradation. Degradation of picric acid was concomitant with stoichiometric release of three moles of nitrite per mole of picric acid. The four picric acid degraders were identified as close relatives of Nocardioides simplex (ATCC 6946) based on their small subunit (16S) rRNA gene sequences.

Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy (soyC) and ferredoxinsoy (soyB) from Streptomyces griseus.

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P-450, designated cytochrome P-450soy. A DNA segment carrying the structural gene encoding P-450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P-450soy was compared with that for P-450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe-4S] ferredoxin (soyB), named ferredoxinsoy, was identified.

Activation and detection of (pro)mutagenic chemicals using recombinant strains of Streptomyces griseus.

Two recombinant strains of Streptomyces griseus have been developed to report on the activation of promutagenic chemicals. This activation is monitored by reversion of the bacterial test strains to a kanamycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol, cis-stilbene, trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such as N-methyl-N'-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).

Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.

Lack of regio- and stereospecificity in oxidation of (+) camphor by Streptomyces griseus enriched in cytochrome P-450soy.

Oxidation of (+) camphor by cytochrome P-450soy-enriched intact cells of Streptomyces griseus resulted in the formation of one major and several minor metabolites. The minor metabolites were identified as 3-endo-hydroxycamphor (2%), 5-endo-hydroxycamphor (7%), 5-exo-hydroxycamphor (9%), 2,5-diketobornane (2%), and camphorquinone (3%). The major metabolite was isolated and conclusively identified as 6-endo-hydroxycamphor (60%). When supplemented with NADPH, spinach ferredoxin:NADP oxidoreductase and spinach ferredoxin, homogeneous preparations of cytochrome P-450soy oxidized camphor to a mixture of 3-endo-, 5-endo-, 5-exo-and 6-endo-hydroxycamphor. The data presented indicates that cytochrome P-450soy resembles its mammalian counterparts in its lack of regio- and stereospecificity in camphor oxidation.

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus.

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.

Primary structure of a 7Fe ferredoxin from Streptomyces griseus.

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy.

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated.

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.

In vitro metabolic transformations of vinblastine: oxidations catalyzed by peroxidase.

Vinblastine is converted to a single major metabolite during in vitro enzymatic oxidations catalyzed by horseradish peroxidase in the presence of hydrogen peroxide. Preparative-scale enzyme incubation permitted the isolation of sufficient amount of the transformation product for complete structural identification and biological evaluation. The metabolite was identified as catharinine (also known as vinamidine) by 1H and 13C NMR and by mass spectrometry. Incubations conducted in H2(18)O-enriched water gave catharinine in which a single atom of 18O was incorporated into the metabolite structure. The labeling experiment provided evidence for an unusual ring-fission pathway by which peroxidase transforms vinblastine to catharinine. Catharinine is 77 times less active than vinblastine when tested in vitro against the human T-cell leukemic cell line (CRFF-CEM).

Microbial enzymes for oxidation of organic molecules.

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.

Xenobiotic oxidation by cytochrome P-450-enriched extracts of Streptomyces griseus.

Crude extracts of Streptomyces griseus grown on soybean flour-enriched medium contain high levels of cytochrome P-450. The cytochrome P-450-enriched fractions, obtained by ammonium sulfate fractionation (30-50% saturation), catalyze the NADPH-dependent oxidation of a variety of xenobiotics when complemented with both spinach ferredoxin:NADP+ oxidoreductase and spinach ferredoxin. Reactions observed are aromatic, benzylic and alicyclic hydroxylations, O-dealkylation, non-aromatic double bond epoxidation, N-oxidation and N-acetylation.

Microbial hydroxylation of 1,4-cineole.

Microorganisms were examined for their potential to hydroxylate the oxygenated monoterpene 1,4-cineole. Using gas chromatography and thin-layer chromatography, screening experiments revealed that hydroxylation at position 2 was the most commonly observed microbial transformation reaction. In most microorganisms, the predominant alcohol metabolite was the 2-endo-alcohol isomer. Preparative-scale incubations were conducted in order to isolate and characterize microbial transformation products by comparison of proton nuclear magnetic resonance, mass spectrometry, and chromatography profiles with those of cineole standards. Streptomyces griseus yielded 8-hydroxy-1,4-cineole as the major hydroxylation product together with 2-exo- and 2-endo-hydroxy-1,4-cineoles.

Microbial transformation of precocene II: oxidative reactions by Streptomyces griseus.

Various species of "Streptomyces," "Aspergillus," "Rhodotorula," "Brevilegnia," "Syncephalastrum," and "Stysanus" were found to transform precocene II to three major metabolites. These major biotransformation products were isolated from a preparative-scale incubation of precocene II with Streptomyces griseus and were conclusively identified as (-)cis- and (+)trans-precocene II-3,4-dihydrodiols and (+)-3-chromenol. 18O2 incorporation studies indicated the involvement of a monooxygenase enzyme system in precocene II transformation by S. griseus. A mechanism is proposed for the formation of (+)-3-chromenol.

Induction of cytochrome P-450 in Streptomyces griseus by soybean flour.

Soybean flour and the isoflavonoid genistein, were found to induce cytochrome P-450 in Streptomyces griseus. The chromophore was found in the 105,000xg supernatant and gave a reduced CO-difference spectrum with an absorption maximum of 448 nm. Almost 70% of the P-450 could be precipitated at 35-45% ammonium sulfate saturation. SDS-gel electrophoresis revealed the presence of a 45,000 dalton polypeptide in extracts induced by either soybean or genistein. S. griseus generated the free isoflavonoids genistein and daidzein when grown on soybean flour medium.

One-electron oxidation of vindoline and 16-O-acetylvindoline catalyzed by peroxidase.

The mechanism of oxidation of the alkaloids vindoline (1) and 16-O-acetylvindoline (1a) was examined by use of the reversible redox cycle of horseradish peroxidase (HRP). Oxidation of 1 by HRP resulted in the formation of the enamine dimer 5. The highly reactive radical cation species 2 is an implied intermediate in the oxidation process. During the reaction, HRP-I was reduced to HRP-II by abstraction of an electron from vindoline. The vindoline radical thus formed eliminates a second electron and a proton to produce a highly reactive iminium derivative which undergoes intramolecular etherification and dimerization. Oxidation of 16-O-acetylvindoline (1a) by HRP-I results in the production of an iminium derivative 3a concomitant with the formation of HRP-II. The iminium 3a was isolated and characterized and was converted into monodeuterated 1a by reduction with NaBD4. The stoichiometry (HRP-II)/(substrate) was determined to be 4.77 +/- 0.17 for vindoline and 2.27 +/- 0.20 for 16-O-acetylvindoline. The enamine dimer also reduced HRP-I to form HRP-II, but the stoichiometry of this reaction was variable.

Biotransformations of 1',2'-dihydrorotenone by Streptomyces griseus.

Dihydrorotenone yields three major products when incubated with growing cultures of Streptomyces griseus. These were isolated by solvent extraction and characterized by spectral methods as 1',2'-dihydro-6abeta-hydroxyrotenone, 1',2'-dihydro-2',6abeta-dihydroxyrotenone, and 1',2'-dihydro-1',6abeta-dihydroxyrotenone.