Carbon Monoxide Difference Spectrum Research Articles

Characterization of the ybdT gene product of Bacillus subtilis: novel fatty acid beta-hydroxylating cytochrome P450.

We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

An unusual cytochrome o'-type cytochrome c oxidase in a Bacillus cereus cytochrome a3 mutant has a very high affinity for oxygen.

Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa3 or caa3. The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied. Respiratory oxidase activities and the levels of cytochromes b and c increased 2.6- to 4.2-fold on transition from exponential growth, in either of two media, to sporulation stage III, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethyl-p-phenylenediamine (TMPD) oxidase activity, consistent with the TMPD- phenotype of strain PYM1. Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron mediator, suggesting that the terminal oxidase receives electrons from a cytochrome c. Carbon monoxide (CO) difference spectra of membranes were obtained using various reductants (ascorbate +/- TMPD, NADH, dithionite) and revealed a haemoprotein resembling cytochrome o'. The CO complex of this cytochrome was photodissociable: the photodissociation spectrum (photolysed minus CO-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the alpha/beta region of the spectrum, consistent with the presence of a cytochrome o'-like pigment. CO recombination occurred at -85 to -95 degrees C. No other haemoproteins showing photoreversible CO binding under these conditions were detected. Evidence that this pigment was the oxidase responsible for substrate oxidation was obtained by photodissociating the CO complex at subzero temperatures in the presence of oxygen; this resulted in faster ligand recombination, attributed to oxygen binding, and extensive oxidation of cytochromes c and b. The oxygen affinity of the oxidase was determined by using the deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissociated oxygen concentration. A single oxidase was revealed with a K(m) for oxygen of about 8 nM; this is one of the highest affinities yet reported for a terminal oxidase.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing.

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.

Two-Step Purification of Cytochrome P-450 from Adult Pig Testis by Isoelectric Focusing

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17a-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.

Symbiotic deficiencies associated with a coxWXYZ mutant of bradyrhizobium japonicum

The terminal oxidase complexes encoded by coxMNOP and coxWXYZ were studied by analysis of mutations in each of the two oxidases. Carbon monoxide difference spectra obtained from membranes of coxMNOP mutant bacteroids were like those obtained for the wild type, whereas bacteroid membranes of a coxWXYZ mutant were deficient in CO-reactive cytochrome b. Experiments involving cyanide inhibition of oxidase activity were consistent with the conclusion that the coxX mutant is deficient in a membrane-associated O2-binding component. The viable cell number (bacteria that could be recultured from crushed nodules) was 20 to 29% lower for the coxX mutant than for the wild-type or the CoxN- strain. In three separate greenhouse studies, nodules of a coxX mutant had significantly lower (28 to 34% less) acetylene reduction rates than the wild-type nodules did, and plants inoculated with a double mutant (coxMNOP coxWZYZ) had rates 30% lower than those of wild-type-inoculated plants.

Stress Survival of a Genetically Engineered Pseudomonas in Soil Slurries: Cytochrome P-450cam-Catalyzed Dehalogenation of Chlorinated Hydrocarbons.

Biological treatment of hazardous chemical wastes has potential as an effective, practical, and economically viable process in above the ground treatment systems that consist of both genetically engineered microorganisms (GEMs) and bioreactors with process control instruments to create ideal conditions for biodegradation. A strain of Pseudomonas putida coexpressing cytochrome P-450cam and luciferase (lux) that provides both the reductive detoxification potential of the hemoprotein and a mechanism for its reduction in the absence of "normal" P-450 redox partners was evaluated for its ability to survive and remain metabolically competent under nutrient stress in soil slurry microcosms. More than 74% of the cells of engineered Pseudomonas were culturable after 7 days of multiple nutrient (C,N,P) starvation. The diagnostic luminescence and carbon monoxide-difference spectra for the two engineered traits could be detected in a significant fraction of the surviving population. The GEM could be revived after repeated desiccation and starvation using Luria broth, benzoate, or citrate as nutrients. Soil slurries inoculated with the GEM transformed hexachloroethane (HCE) to tetrachloroethylene (tetraCE) 8-10-fold faster than uninoculated slurries. The GEM also transformed the insecticide, gamma-HCH (gamma-3,4,5,6-hexachlorocyclhexene), to gamma-3,4,5,6-tetrachlorocyclohexene (gammatetraCH) in soil slurries under subatmospheric conditions. These results indicate that GEMs can be constructed with broad substrate range detoxification catalysts such as cytochrome P-450 for remediation.

Mechanism of fluconazole resistance in Candida krusei.

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14alpha-demethylase in this organism. The 14alpha-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by fluconazole than was 14alpha-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of C. albicans. Thus, there are significant qualitative differences between the 14alpha-demethylase of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14alpha-demethylase to inhibition by this drug.

Glyphosate Is an Inhibitor of Plant Cytochrome P450: Functional Expression ofThlaspi arvensaeCytochrome P45071B1/Reductase Fusion Protein inEscherichia coli

Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5′enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed. Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system forThlaspi arvensaeCYP71B1 inEscherichia coli.CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) fromCatharanthus roseus.CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene. The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand. A Ksof 60 μM was observed and an IC50of 12 μM was observed for glyphosate inhibition of CYP71B1 activity. The implications of these results are discussed.

Degradation of morpholine by an environmental Mycobacterium strain involves a cytochrome P-450.

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C-N bond of the morpholine ring.

Changes of Insecticide Resistance Levels and Detoxication Enzymes Following Insecticide Selection in the German Cockroach,Blattella germanica(L.)

Insecticide resistance in the German cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism. Changes in metabolic enzymes following insecticide selection of a hybrid German cockroach strain (JM) were investigated. The JM strain originated from matings of the Munsyana (multiple resistance) strain and Johnson Wax (susceptible) strain. Two subpopulations of the JM strain were selected over three generations: one with a pyrethroid insecticide (cypermethrin) and one with an organophosphate insecticide (chlorpyrifos). Significant increases in resistance levels were observed in the first generation following selection. These trends continued for three generations and resistance levels to each insecticide approached those of the original Munsyana strain by the third generation of selection. Cypermethrin selected for cross-resistance to chlorpyrifos; however, chlorpyrifos selected for negative cross-resistance to cypermethrin. Increased body weight was weakly but positively correlated with increases in insecticide resistance to each insecticide. Examinations of metabolic enzymes indicated no change in total esterase (1-NA, 2-NA, and PNPA hydrolysis) or glutathione S-transferase (DCNB and CDNB conjugation) activity. Native PAGE of esterases identified one electromorph (E2) which stained more intensely following chlorpyrifos selection and which was inhibited by both chlorpyrifos and chlorpyrifosoxon. Examination of cytochromes P450 by carbon monoxide difference spectra indicated that each insecticide selected for increases in total cytochrome P450 content. However,in vitrodemethylation and Western analysis results suggest that different cytochrome P450 isoforms were selected by each insecticide.

Purification and characterization of a cytosolic cytochrome P450 from the yeast Trichosporon cutaneum

A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns. This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8%. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa. The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum. This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH. The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum.

Expression of CYP71B7, a Cytochrome P450 Expressed Sequence Tag fromArabidopsis thaliana

The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plantArabidopsis thalianahas identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designatedCYP71B7in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene inA. thaliana.Northern hybridization analysis indicated that maximal expression ofCYP71B7occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that theCYP71B7gene was likely to exist as a single copy in the genome ofA. thaliana. CYP71B7was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressingCYP71B7were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressingCYP71B7or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH–ferricytochrome P450 reductase (CPR) activity. The data suggest that thisA. thalianacyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii.

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

The Mutation T315A in Candida albicans Sterol 14α-Demethylase Causes Reduced Enzyme Activity and Fluconazole Resistance through Reduced Affinity

Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.

Investigation of Cytochrome P450 Mediated Benzo(a)pyrene Hydroxylation inAspergillus fumigatus

Microsomal fractions of Aspergillus fumigatus exhibited a reduced carbon monoxide difference spectrum with a maximum at 448 nm and a substrate-induced Type I spectrum on addition of benzo(a)pyrene. Metabolism of benzo(a)pyrene, as measured using the aryl hydrocarbon hydroxylase assay, was found to be P450-dependent and activity was observed to have a K, of 82 p~ and V,,, of 33.3 pmol min-' mg-' protein. P450 productivity was investigated during growth from spore inocula and observed to be constant until the station- ary phase, where the specific content declined to undetectable levels. The specific content of P450 was found to increase in higher concentrations of glucose.

Peroxidative reactions of diversozymes 1

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive beta-cleavage to give hydrocarbons and aldehydic acids. One of these products, trans-4-hydroxynonenal, inactivates P450, particularly alcohol-inducible 2E1, in what may be a negative regulatory process. Although a P450 iron-oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron-peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.

Evidence for cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium.

The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448-450 and 452-454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH dependent and inhibited by carbon monoxide. Km values for activity showed a difference between the cellular fractions with a Km of 89 microM for microsomal P-450 and 400 microM for cytosolic P-450. The Vmax values observed were 0.83 nmol min-1 (nmol microsomal P-450)-1 and 0.4 nmol min-1 (nmol cytosolic P-450)-1. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.

Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium

The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K m values for activity showed a difference between the cellular fractions with a K m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V max values observed were 0.83 nmol min − (nmol microsomal P-450) −1 and 0.4 nmol min −1 (nmol cytosolic P-450) −1. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.

Evidence That Aspartic Acid 301 Is a Critical Substrate-Contact Residue in the Active Site of Cytochrome P450 2D6

Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.