Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cytochrome P450 monooxygenase commonly used for negative selection in transgenic plants causes growth anomalies by disrupting brassinosteroid signaling

Background  

Cytochrome P450 monooxygenases form a large superfamily of enzymes that catalyze diverse reactions. The P450 _SU1 gene from the soil bacteria Streptomyces griseolus encodes CYP105A1 which acts on various substrates including sulfonylurea herbicides, vitamin D, coumarins, and based on the work presented here, brassinosteroids. P450 _SU1 is used as a negative-selection marker in plants because CYP105A1 converts the relatively benign sulfonyl urea pro-herbicide R7402 into a highly phytotoxic product. Consistent with its use for negative selection, transgenic Arabidopsis plants were generated with P450 _SU1 situated between recognition sequences for FLP recombinase from yeast to select for recombinase-mediated excision. However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene

Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O₂, this polypeptide (cytochrome P-450_BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450_BM-3 from B. megaterium and putative P-450_BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450_BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS Sodium Dodecylsulfate - PAGE Polyacrylamide Gel Electrophoresis - HPLC High Performance Liquid Chromatography     

Induction of cytochrome P-450-dependent sulfonylurea metabolism in Streptomyces griseolus

Inducible cometabolism of several sulfonylurea herbicides by Streptomyces griseolus has been shown to occur by hydroxylation, O-dealkylation, or deesterification reactions. Only after growth of the bacterium in the presence of sulfonylurea did cell-free extracts exhibit NAD(P)H-dependent sulfonylurea metabolism. These extracts were shown to contain elevated levels of soluble cytochrome P-450 and exhibit sulfonylurea induced difference spectra consistent with binding of substrate to cytochrome(s) P-450. These results establish the presence of an inducible cytochrome P-450-dependent sulfonylurea metabolizing system in S. griseolus.     

Identification of a cytochrome P450 hydroxylase, CYP81A6, as the candidate for the bentazon and sulfonylurea herbicide resistance gene, Bel, in rice

Bentazon and sulfonylureas have been used for selective control of broadleaf weeds and sedges in rice fields for more than 20 years. A bentazon and sulfonylurea susceptible mutant, bel, was previously identified for the purpose of allowing these herbicides to be used for removing false hybrids from hybrid rice. While this mutation has been used successfully in rice breeding, the genetic nature of bel is not known. Using 1,776 susceptible plants from a population of 10,000 F₂ individuals, we constructed a fine map for the Bel locus and delimited it to a 36-kb DNA fragment between two restriction fragment length polymorphism markers, L5 and P17. Bioinformatic analysis indicated that there are five genes within this interval, an ethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genes designated as CYP81A5, CYP81A6, CYP81A7, and CYP81A8. Comparative sequencing could not find any differences in the coding regions of the OsER33, CYP81A5, CYP81A7, and CYP81A8 genes between the mutant bel and its wild-type progenitor W6154S, but did identify a single base guanine deletion at position +1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contains an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylurea herbicides in rice. A gene-specific cleaved amplified polymorphic sequence marker and tightly linked flanking markers were developed that will be very useful for selection of the bel allele when transferred to photoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breeding programs.     

Function and regulation of cytochrome P-450 in alkane-assimilating yeast

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO₂=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO₂ of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.Abbreviation cyt cytochrome     

Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

The biochemical genetics of permethrin resistance in the Learn-PyR strain of house fly

Permethrin resistance in the Learn-PyR strain of house fly was examined in four genetically derived substrains, each being homozygous for a different resistant autosome of the Learn-PyR strain. The resistance of these derivative strains was characterized toxicologically and biochemically. The relative levels of resistance to permethrin conferred by each autosome were 5>3>1>2. Three factors were associated with resistance: (1) increased mixed-function oxidase (MFO) activity associated with elevated levels of cytochrome P-450, cytochrome b₅, and NADPH-cytochrome c reductase (P-450 reductase) activity; (2) target-site insensitivity (kdr); and (3) decreased cuticular penetration. Permethrin resistance factors on chromosome 1 consisted of a piperonyl butoxide (PB)-suppressible mechanism correlated with increased levels of cytochromes P-450 and b₅; on chromosome 2, a PB-suppressible mechanism associated with elevated amounts of cytochrome P-450; on chromosome 3, decreased cuticular penetration, kdr, and increased amounts of P-450 reductase activity; and on chromosome 5, a largely PB-suppressible mechanism correlated with elevated levels of cytochrome P-450 and P-450 reductase activity.     

The oxidative metabolism of arachidonic acid by purified cytochromes P-450

Arachidonic acid is catalytically oxidized using either of two types of purified cytochrome P-450 reconstituted with the purified flavo-protein, NADPH-cytochrome P-450 reductase. The reaction is dependent on the presence of cytochrome P-450, NADPH, and oxygen. The patterns of products formed are unique for the type of cytochrome P-450 used. This suggests an enzyme-directed specificity of the site of attack on the unsaturated fatty acid by the hemeprotein. Additional experiments show a possible role for cytochrome b₅ since the addition of purified cytochrome b₅ enhances the rate of metabolism of arachidonic acid 2 to 3 fold.     

The carbon monoxide- and oxygen-reacting haemoproteins of Streptomyces clavuligerus: cytochrome aa 3 is the predominant terminal oxidase of the respiratory chain

The nature of the carbon monoxide- and oxygen-reacting haemoproteins in the respiratory chain of the filamentous antibiotic-producing bacterium Streptomyces clavuligerus has been investigated. CO-difference (i.e. CO+ reduced minus reduced) spectra of intact cells showed the presence of cytochrome aa ₃, a CO binding b-type cytochrome, and a pigment resembling cytochrome d. In addition, cells that were approaching the end of the growth phase showed the presence of cytochrome P450: this pigment was undetectable in cells harvested early in the growth cycle. High speed centrifugation of cell-free extracts prepared from cells broken by sonication showed that cytochrome aa ₃ was tightly membrane-bound and that cytochrome P450 was soluble. Inhibition of oxygen uptake rates of cells by cyanide indicated that one component, which showed 50% inhibition at 2–4 mM CN^–, was acting as major terminal oxidase: this was observed in cells harvested from all stages of growth. Photodissociation (i. e. photolysed, CO reduced minus CO reduced) spectra at-118°C, in the absence of oxygen, showed cytochrome aa ₃ to be the sole photolysable CO-reacting haemoprotein. At higher temperature (-87°C), in the presence of oxygen, cytochrome aa ₃ formed a complex with oxygen that could not be photolysed by similar intensities of light. By raising the temperature to-43°C, the oxidation of c-type cytochromes was observed. It is concluded that cytochrome aa ₃ is the predominant terminal oxidase in S. clavuligerus and that the other CO reacting haemoproteins, of unknown function, are unlikely to be oxidases.     

Different requirement for cytochrome b5 in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450

The role of cytochrome b₅ in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450_PB and P-450_MC) purified from rat liver microsomes. The O-deethylation by P-450_PB absolutely required the presence of cytochrome b₅, whereas the same reaction catalyzed by P-450_MC did not require cytochrome b₅. These effects of cytochrome b₅ on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b₅. On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b₅. These observations suggest that the requirement for cytochrome b₅ in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b₅ localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken.     

Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus.

Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al. [D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6:1-11, 1987]). Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1. Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida. Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.     

Alkaloid metabolism by cytochrome P-450 enzymes in Drosophila melanogaster

The cytochrome P-450 family of enzymes is the primary means of foreign compound detoxification in virtually all organisms. Cytochrome P-450s have been strongly implicated in the metabolism of cactus alkaloids, and consequently, the observed patterns of host plant utilization by cactophilic species of Drosophila in the Sonoran Desert. The current study looked for evidence of alkaloid-metabolizing P-450 enzymes in a non-cactophilic species, D. melanogaster. The results of in vitro metabolism assays indicate the presence of a phenobarbital-inducible P-450 in adult D. melanogaster which is capable of metabolizing alkaloids. P-450 quantification data suggest that the enhanced level of metabolism is not the result of an overall increase in total P-450 content. Results from larval viability and adult longevity studies indicate that D. melanogaster's in vitro activity does not produce an enhanced in vivo tolerance of alkaloids.     

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.     

Differential stereoselectivity of cytochromes P-450b and P-450c in the formation of naphthalene and anthracene 1,2-oxides. The role of epoxide hydrolase in determining the enantiomer composition of the 1,2-dihydrodiols formed

As is the case for cytochrome P-450c, arene 1,2-oxides have been identified as initial metabolites when naphthalene and anthracene are oxidized by cytochrome P-450b in a highly purified, reconstituted system. Overall rates of metabolism by cytochrome P-450b are greater than 3-fold and greater than 50-fold lower than the respective rates of metabolism by cytochrome P-450c. For both hydrocarbons, the (-)-(1S,2R)-oxide predominates (74%) with cytochrome P-450b as the terminal oxidant, based on trapping the labile arene oxides as N-acetyl-L-cysteine S-conjugates of known absolute configuration. This result is in marked contrast to data obtained with cytochrome P-450c where the (+)-(1R,2S)-oxides predominate (73-greater than 95%). In the absence of added epoxide hydrolase, the metabolically formed arene oxides rapidly isomerize to phenols. Addition of increasing amounts of epoxide hydrolase to the incubation medium results in the formation of trans-1,2-dihydrodiols at the expense of phenols from the common arene oxide intermediates. Evaluation of the kinetic parameters (Km and kcat) for the hydration of the (+)- and (-)-enantiomers of both arene oxides by epoxide hydrolase has indicated that the (+)-(1R,2S)-enantiomers exhibit lower values of Km (approximately 1 microM) whereas the values of kcat are similar for both enantiomers of a given arene oxide. These parameters have allowed construction of a mathematical model which predicts the enantiomer composition of the dihydrodiols formed from naphthalene in reconstituted systems containing specific epoxide hydrolase concentrations. The data reported argue against a selective functional coupling mechanism between cytochrome P-450c and epoxide hydrolase in the metabolism of naphthalene and anthracene to the 1,2-dihydrodiols.