Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes

According to Ohba et al. (Ohba, M., Sato, R., Yoshida, Y., Nishino, T. and Katsuki, H. (1978) Biochem. Biophys. Res. Commun. 85, 21-27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30-14C]lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol). It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14 alpha-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.     

Oxidative C4-demethylation of 24-methylene cycloartanol by a cyanide-sensitive enzymatic system from higher plant microsomes

Microsomes isolated from corn embryos (Zea mays) were shown to catalyse the C-4 monodemethylation of 28-[3H],24-methylene cycloartanol 1, leading to the corresponding 4 alpha-methyl sterol, cycloeucalenol 5. An enzymatic assay has been developed for the 4,4-dimethyl sterol 4-demethylase in higher plants. The demethylation process was shown to involve a 4-methyl, 4-hydroxymethyl derivative 2 which can be considered as the immediate metabolite of 1 by the 4-methyl oxidase. Compound 2 is further metabolized into 5 through a 4-methyl-4-carboxylic acid 3 and a 3-keto-4 alpha-methyl intermediate 4 which were identified. The conversion of 1 into 5 requires NADPH and molecular oxygen. The initial oxidative step was strictly dependent upon molecular oxygen, NADPH or NADH, and strongly inhibited by cyanide, whereas the overall process was completely insensitive to CO and to specific inhibitors of cytochrome P-450. It is concluded that in Zea mays microsomes, the C-4 demethylation of 1 results from a multistep process involving a terminal oxygenation system sensitive to cyanide which is distinct from cytochrome P-450 and in particular from that involved in the 14 alpha-demethylation of obtusifoliol.     

Purification and characterization of intact cytochrome b5 from yeast microsomes

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.     

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes.

The involvement of cytochrome b5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or delta-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b5, but those from delta-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b5 and catalyzed the desaturation. The cytochrome b5 content in microsomes from mutant Ole 3 could be varied by changing the delta-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b5 content was increased. The antibody to yeast cytochrome b5, but not the control gamma-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b5 is actually involved in the desaturase system of yeast microsomes. The lack of desaturase activity in mutant Ole 3 grown with Tween 80 plus ergosterol seems to be due to the absence of cytochrome b5 in microsomes, whereas the genetic lesion in mutant E5 appears to be located at ther terminal desaturase.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Alpha-hydroxylation of lignoceroyl-CoA by a cyanide-sensitive oxygenase in rat brain microsomes

The enzymatic mechanism of alpha-hydroxylation of lignoceroyl-CoA, an intermediate in the synthesis of hydroxyceramide, was studied. In the presence of NADPH, sphingosine and microsomes from 20-day-old rat brain, 14C from [1-14C]lignoceroyl-CoA was incorporated into hydroxyceramide. Activity was linear with time (up to 40 min) and with protein (up to 0.8 mg). The apparent Km for lignoceroyl-CoA was about 10 microM. NADPH was a more efficient electron donor than NADH. Oxygen was required for activity, which increased linearly up to 20% O2. In 5 and 10% oxygen, the reaction was inhibited by 0.1 mM cyanide and by electron transfer chain inhibitors, cytochrome c, ferricyanide, menadione, and p-chloromercuriphenyl sulphonate; CO and SKF-525A had no effect. Moreover none of the inhibitors affected the formation of hydroxyceramide. Lignoceroyl-CoA alpha-hydroxylase appears to be an oxygenase requiring NADPH and oxygen, which involves cyanide-sensitive enzyme.     

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.     

Involvement of cytochrome P450 and the flavin-containing monooxygenase(s) in the sulphoxidation of simple sulphides in human liver microsomes

This study was conducted to examine the involvement of cytochrome P450 (CYP450) and the flavin-containing monooxygenase (FMO) in the sulphoxidation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) in human liver microsomes from a phenotypic CYP2D6 extensive metabolizer. Human liver microsomes catalyzed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides. Lineweaver-Burk plots for the sulphoxidation of EMS in human liver microsomes indicated that the apparent K(m) and V(max) were 1.53 +/- 0.07 mM and 1.11 +/- 0.25 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of CPMS were 0.17 +/- 0.05 mM and 1.41 +/- 0.16 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of DPS were 0.10 +/- 0.01 mM and 1.08 +/- 0.05 nmoles/mg protein/min, respectively. Methimazole noncompetitively inhibited the sulphoxidation of EMS, CPMS and DPS by human liver microsomes with K(i) values of 8.6 +/- 0.6, 5.7 +/- 0.4 and 6.6 +/- 0.5 mM, respectively. SKF525A noncompetitively inhibited the sulphoxidation of CPMS and DPS by human liver microsomes with K(i) values of 6.6 +/- 0.4 and 0.40 +/- 0.1 mM, respectively. The results suggest that FMO is involved in the sulphoxidation of EMS, CPMS and DPS while CYP450 is involved in the sulphoxidation of CPMS and DPS in human liver microsomes.     

The 14 alpha-demethylation of obtusifoliol by a cytochrome P-450 monooxygenase from higher plants' microsomes

Microsomes isolated from corn embryos (Zea mays) can demethylate the 14 alpha-methyl group of obtusifoliol 2. An enzymatic assay has been developed for obtusifoliol 14 alpha-methyl-demethylase in higher plants. The enzymatic reaction was shown to occur sequentially, converting obtusifoliol 2 to 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-3 beta-ol 4 via the trienol 4 alpha-methyl-5 alpha-ergosta-8,14,24(28)-trien-3 beta-ol 3 which was thoroughly identified. This enzymatic reaction is dependent of NADPH and molecular oxygen. It is inhibited by CO, menadione and specific inhibitors of cytochrome P-450, the CO inhibition being partially reversed by light. It is concluded that in Zea mays microsomes, obtusifoliol is demethylated at C-14 by a cytochrome P-450 containing monooxygenase system.     

OnpA, an unusual flavin-dependent monooxygenase containing a cytochrome b(5) domain

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b(5) domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b(5) domain were associated with binding of the heme, and the cytochrome b(5) domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b(5) domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b(5) and flavin-dependent monooxygenases.