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

Trower, Michael K.; Sariaslani, F. Sima; O’Keefe, Daniel P.

doi:10.1128/jb.171.4.1781-1787.1989

Cited by 45 publications

(30 citation statements)

References 37 publications

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“…Soybean flour-glycerol and sporulation broth (no. 5; American Type Culture Collection) media used for cultivation of S. griseus (ATCC 13273) and the general fermentation protocol have been described before (25,36). Cells were harvested by centrifugation (8,000 x g for 30 min at 4°C), washed three times with 100 mM sodium phosphate (pH 7.4) buffer containing 1 mM dithiothreitol (DTT) and 0.1 mM EDTA, and stored at -80°C until required.…”

Section: Methodsmentioning

confidence: 99%

“…Adrenodoxin was kindly provided by Lawrence Vickery (University of California, Irvine). Cytochrome P-450soy and the 7Fe ferredoxin were purified from S. griseus cells as described previously (33,36). Other materials used in this study were of the finest commercial grade available.…”

Section: Methodsmentioning

confidence: 99%

“…This enzymatic system also mimics rat liver microsomes in activating a variety of promutagenic chemicals (27). We have previously reported the isolation and characterization of the cytochrome P-450S.Y (36) and S. griseus 7Fe ferredoxin (33,34) a soybean flour-enriched medium. In addition to cytochrome P-450Soy and ferredoxin, in vitro reconstitution experiments have indicated a requirement for the presence of a ferredoxin reductase to provide reducing power from NAD(P)H to the cytochrome P-450 component.…”

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

et al. 1991

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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 Mg21 ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotidecontaining 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-450s,y, the ferredoxin reductase mediates 0 dealkylation of 7-ethoxycoumarin.Microbial cytochrome P-450 systems are usually multicomponent systems and require the presence of a ferredoxin reductase and a ferredoxin to couple electron flow from NAD(P)H to the terminal cytochrome P-450 component. To date, of the microbial cytochrome P-450 systems reported, only the barbiturate-induced cytochrome P-450 system of Bacillus megaterium (ATCC 14581) is self-sufficient and does not require the intermediacy of other redox components (18). In contrast with the cytochromes P-450 and ferredoxins, only a few ferredoxin reductases of microbial cytochrome P-450 systems have been purified and characterized because of their unstable nature and the relatively low level of expression. These examples include ferredoxin reductases of Pseudomonas putida (PA450cam and P-4501j.) (14,21,22,38,39), B. megaterium (P-450meg; ATCC 13368) (2, 9), and Saccharopolyspora erythraea (31). In the cytochrome P-450cam system, the putidaredoxin reductase level is about eight times less than that of cytochrome P-450cam and putidaredoxin (21,22). The ferredoxin reductase of the cytochrome P-450 system of another actinomycete, a Nocardia sp. (5), is also extremely unstable and was only partially purified.Previous studies in this laboratory have indicated that the soybean flour-induced cytochrome P-450S.Y from Streptomyces griseus resembles its mammalian counterparts in oxidizing a diverse array of xenobiotics (24,26,28,29,35). The reactions catalyzed include aromatic, cyclic, and aliphatic hydroxylation; 0, S, and N oxidations; C-C fission; epoxidation; and 0 and N dealkylations. This enzymatic system also mimics rat liver microsomes in activating a variety of promutagenic chemicals (27). We have previously reported the isolation and characterization of the cytochrome P-450S.Y (36) and S. griseus 7Fe ferredoxin (33, 34) from extracts of S. griseus cells grown in * Corresponding author.t Present address:

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

et al. 1991

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“…For reconstitution of the P-450mel activity in vitro, we used the ferredoxin and ferredoxin-NADP ϩ reductase from spinach, as was successfully used for reconstitution of a soluble cytochrome P-450 soy from S. griseus (20). Incubation of the reconstituted system with THN gave two products, as analyzed by HPLC (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of Hexahydroxyperylenequinone Melanin via Oxidative Aryl Coupling by Cytochrome P-450 in Streptomyces griseus

et al. 2005

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Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NADP ؉ reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter.The genus Streptomyces comprises gram-positive, soil-dwelling, filamentous bacteria with a complex life cycle similar to that of fungi. In addition to the complex morphological differentiation, Streptomyces is also characterized by its ability to produce a wide variety of secondary metabolites, including pharmaceutically useful compounds, such as antibiotics, antitumor agents, and immunosuppressants (3). Melanins, which are high-molecular-weight dark-brown to black pigments, are secondary metabolites produced by diverse species of streptomycetes and are used for the taxonomy of this genus (24,25). Various organisms synthesize a so-called dihydroxyphenylalanine (DOPA) melanin, which is a nonenzymatically or laccase-mediated polymerized product from DOPA. DOPA is formed from tyrosine by the oxidative action of tyrosinases. The representative of the tyrosinases responsible for the formation of DOPA melanin in Streptomyces is MelC2 (5), which is a copper-containing monooxygenase that catalyzes the o-hydroxylation of monophenols and the oxidation of o-diphenols to yield o-quinones using molecular oxygen (2). Because tyrosine occurs universally in living organisms, tyrosinases are also responsible for browning in plants and melanization in animals (2).We previously found that a mutation in rppA, encoding a type III polyketide synthase (PKS), caused the host, Streptomyces griseus, to show an "albino" phenotype (8). Although S. griseus produces DOPA melanin, its production is apparent only ...

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“…The tree also comprises the CYPome of Mycobacterium tuberculosis (20 P450s in green with prefix 'Mtb') [36], Streptomyces coelicolor A(3)2 (18 P450s in violet with prefix 'Sc') [37], Rhodopedomonas palustris (nine P450s in pink with prefix 'Rp') and Novosphingobium aromaticivorans DSM 12444 (14 P450s in grey with prefix 'Na') (http://www.cyped.uni-stuttgart.de). The well characterized bacterial P450s, CYP176A1_P450cin from Citrobacter braakii (AF456128_1) [38], CYP107H1_P450BioI from Bacillus subtilis (NP_390897.1) [39], CYP106A2_Bm from Bacillus megaterium (CAA79985) [40], CYP119_Ss from Sulfolobus acidocaldarius DSM 639 (1F4T_A) [41], CYP107A1_eryF from Saccharopolyspora erythraea NRRL 2338 (1Z8O_A) [42], CYP101_camC from Pseudomonas putida (1RF9_A) [43], CYP266A3_tdtD from Pseudomonas diterpeniphila (AF274704_2) [44], CYP167A1_epoK from Sorangium cellulosum (AF217189_9) [45], P-450soy (CAA45146) [46] and P450-SU1 (CYP105A1) [47] from Streptomyces griseolus (AAA26823), CYP210A1_SpiL from Polyangium cellulosum (CAD43453) [48] and Leu_Orf24 from Chondromyces crocatus (CAQ18837) [49] (all shown in brown), are also included. The relatedness of the S. cellulosum So ce56 clustered with the other P450s are shown.…”

Section: Comparison Of the Cypomes Of Spore-forming Myxobacteriamentioning

confidence: 99%

Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

et al. 2011

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a b s t r a c tThe exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed.

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

Cited by 45 publications

References 37 publications

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

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

Biosynthesis of Hexahydroxyperylenequinone Melanin via Oxidative Aryl Coupling by Cytochrome P-450 in Streptomyces griseus

Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

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