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.

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.

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).

Formation of a reactive iminium derivative by enzymatic and chemical oxidations of 16-O-acetylvindoline.

16-O- Acetylvindoline (1a) was oxidatively transformed into an iminium derivative (2a) by copper oxidases (laccase and human ceruloplasmin), an unknown enzyme system(s) of Streptomyces griseus, and the chemical oxidizing agent 2,3-dichloro-5,6- dicyano -1,4-benzoquinone ( DDQ ). The iminium derivative (2a) was isolated from enzymatic and chemical oxidation mixtures and was identified by spectral and chemical techniques. Reduction of the iminium compound with sodium borodeuteride provided monodeuterated 16-O- acetylvindoline (1b) as the sole product. Mass spectral analysis indicated that the deuterium atom was introduced into position C-3 of the piperidine portion of the alkaloid structure. The location and stereochemistry of the deuterium atom were confirmed by high-field 1H and 2H NMR analyses of the deuterated product to be in the 2H alpha orientation. Hydrolysis of the 16-O-acetyl functional group from the iminium derivative (2a) resulted in the production of a previously identified dimer (5), which forms by intramolecular etherification through the reactive enamine (3). The iminium derivative (2a) reacts with cyanide to provide complex mixtures of products, one of which was identified by mass spectrometry as a cyanide addition product. The results confirm the existence of a reactive iminium intermediate formed by all of the biochemical and chemical systems examined.

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).

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.

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.

Microbial Transformations of Natural Antitumor Agents: Products of Rotenone and Dihydrorotenone Transformation by Cunninghamella blakesleeana.

Various species of Absidia, Aspergillus, Cunninghamella, Trichothecium, Penicillium, and Phanerochaete were found to transform rotenone to one or more metabolites. Two biotransformation products were isolated from a preparative-scale incubation of rotenone with Cunninghamella blakesleeana and identified as 1',2'-dihydro-1',2'-dihydroxyrotenone and 3'-hydroxyrotenone (amorphigenin). The catalytic reduction of the isopropylene side chain of rotenone resulted in the formation of 1',2'-dihydrorotenone. The latter was transformed by C. blakesleeana to 2'-hydroxy-1',2'-dihydrorotenone.

Novel Biotransformations of 7-Ethoxycoumarin by Streptomyces griseus.

Biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of two metabolites which were isolated and identified as 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin. A novel series of biotransformation reactions is implicated in the conversion of the ethoxycoumarin substrate to these products, including O-deethylation, 6-hydroxylation to form a 6,7-dihydroxycoumarin catechol, and subsequent O-methylation. Either 7-hydroxycoumarin or 6,7-dihydroxycoumarin was biotransformed to 7-hydroxy-6-methoxycoumarin by S. griseus. Trace amounts of the isomeric 6-hydroxy-7-methoxycoumarin were detected when 6,7-dihydroxycoumarin was used as the substrate. Efforts to obtain a cell-free catechol-O-methyltransferase enzyme system from S. griseus were unsuccessful. However, [methyl-C]methionine was used with cultures of S. griseus to form 7-hydroxy-6-[C]methoxycoumarin.

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.

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.

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.

Oxidation of rotenone by Polyporus anceps laccase.

The extracellular laccase produced by Polyporus anceps transforms rotenone to a single, more polar product. This transformation occurs in incubation mixtures containing chlorpromazine, laccase, and rotenone where rotenone serves as a pseudosubstrate for the enzyme. Chlorpromazine, the true substrate, serves as a cycling redox component of the system forming a radical-cation species that abstracts an electron from rotenone in the oxidation process. Physicochemical properties of the product were determined on an analytically pure sample obtained by preparative hplc. High resolution ms, high-field pmr and cmr, uv, and optical rotation analyses indicated that rotenone had been transformed to 6a beta, 12a beta-rotenolone by P. anceps laccase.

Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor.

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by omega-oxidation of the terminal methyl group, side-chain beta-oxidation to 4-phenylbutyrate, both beta- and alpha-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.

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.

Control of isocitrate lyase in Nocardia salmonicolor (NCIB9701).

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.

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.