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Sample records for cytochrome b5 reductase

  1. Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver.

    PubMed

    Arinç, E; Cakir, D

    1999-02-01

    Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme

  2. Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5.

    PubMed

    Kurian, Joseph R; Chin, Nathaniel A; Longlais, Brett J; Hayes, Kristie L; Trepanier, Lauren A

    2006-10-01

    Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of

  3. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction

    PubMed Central

    Sacco, James C.; Trepanier, Lauren A.

    2010-01-01

    Objectives NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Methods Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semi-quantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Results Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06–1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (p < 0.0001, r = 0.42; p = 0.01, r = 0.23, respectively), and expression of both proteins correlated with one another (p < 0.0001; r = 0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. Conclusion These studies indicate that while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction. PMID:19997042

  4. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  5. Structure of Physarum polycephalum cytochrome b 5 reductase at 1.56 Å resolution

    PubMed Central

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-01-01

    Physarum polycephalum cytochrome b 5 reductase catalyzes the reduction of cytochrome b 5 by NADH. The structure of P. polycephalum cytochrome b 5 reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b 5 reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data. PMID:17401193

  6. Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.

    PubMed

    Maeda, Shintaro; Kobori, Hiroki; Tanigawa, Minoru; Sato, Katsuya; Yubisui, Toshitsugu; Hori, Hiroshi; Nagata, Yoko

    2015-07-01

    For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 μM and 160 μmol min(-1) mg(-1), respectively, for ferricyanide and 328 μM and 500 μmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA. PMID:25829149

  7. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans. PMID:26806391

  8. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.

  9. Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Bork, Bettina; Bittner, Florian; Mendel, Ralf; Clement, Bernd

    2016-10-01

    The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.

  10. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours.

    PubMed Central

    Marín, A.; López de Cerain, A.; Hamilton, E.; Lewis, A. D.; Martinez-Peñuela, J. M.; Idoate, M. A.; Bello, J.

    1997-01-01

    The level of expression of enzymes that can activate or detoxify bioreductive agents within tumours has emerged as an important feature in the development of these anti-tumour compounds. The levels of two such reductase enzymes have been determined in 19 human non-small-cell lung tumours and 20 human breast tumours, together with the corresponding normal tissue. DT-diaphorase (DTD) enzyme levels (both expression and activity) were determined in these samples. Cytochrome b5 reductase (Cytb5R) activity was also assessed. With the exception of six patients, the levels of DTD activity were below 45 nmol min(-1) mg(-1) in the normal tissues assayed. DTD tumour activity was extremely variable, distinguishing two different groups of patients, one with DTD activity above 79 nmol min(-1) mg(-1) and the other with levels that were in the same range as found for the normal tissues. In 53% of the lung tumour samples, DTD activity was increased with respect to the normal tissue by a factor of 2.4-90.3 (range 79-965 nmol min[-1] mg[-1]). In 70% of the breast tumour samples, DTD activity was over 80 nmol min(-1) mg(-1) (range 83-267 nmol min[-1] mg[-1]). DTD expression measured by Western blot correlated well with the enzyme activity measured in both tumour and normal tissues. The levels of the other reductase enzyme, Cytb5R, were not as variable as those for DTD, being in the same range in both tumour and normal tissue or slightly higher in the normal tissues. The heterogeneous nature of DTD activity and expression reinforces the need to measure enzyme levels in individual patients before therapy with DTD-activated bioreductive drugs. Images Figure 1 Figure 2 PMID:9328153

  11. NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I.

    PubMed

    Percy, M J; Aslan, D

    2008-10-01

    The development of cyanosis at birth, the so-called blue baby syndrome, alerts paediatricians to the presence of congenital heart disease. In rare cases where the arterial blood gas analysis is normal the cyanosis is a consequence of methaemoglobinaemia. There are three distinct origins of methaemoglobinaemia; the presence of a haemoglobin variant, environmental toxicity and deficiency of cytochrome b5 reductase (cb(5)r). Two children born to two sets of first-degree related parents were cyanotic from birth. Differential diagnosis eliminated cardiac and pulmonary abnormalities. Measurement of methaemoglobin levels confirmed recessive congenital methaemoglobinaemia (RCM) and treatment with ascorbic acid was commenced. In the absence of neurological defects, type I disease was diagnosed. Sequence analysis of CYB5R3 revealed two different missense mutations (one which is novel, Ile85Ser) in the two families. Neither of the mutations was located in the FAD or the NADH binding sites of cb(5)r, thus supporting a diagnosis of type I disease. PMID:18820099

  12. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

    PubMed Central

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  13. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene.

    PubMed

    Stiborová, Marie; Indra, Radek; Moserová, Michaela; Frei, Eva; Schmeiser, Heinz H; Kopka, Klaus; Philips, David H; Arlt, Volker M

    2016-08-15

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  14. Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous

    PubMed Central

    Gutiérrez, María Soledad; Rojas, María Cecilia; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2015-01-01

    The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. PMID:26466337

  15. Study of the Individual Cytochrome b5 and Cytochrome b5 Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain*

    PubMed Central

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R.; Battaile, Kevin P.; Lovell, Scott; Benson, David R.; Zhu, Hao

    2010-01-01

    NADH cytochrome b5 oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b5 (b5), CHORD-SGT1 (CS), and cytochrome b5 reductase (b5R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b5 and b5R domains (Ncb5or-b5 and Ncb5or-b5R, respectively) and compared them with human microsomal b5 (Cyb5A) and b5R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b5 reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His89 and His112, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b5 family shown to have such a heme environment. Like other b5 family members, Ncb5or-b5 has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b5 differs from Cyb5A with respect to location of the second heme ligand (His112) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b5R to Ncb5or-b5 is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b5 and b5R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b5R domains suggest that the CS domain facilitates docking of the b5 and b5R domains. Trp114 is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b5R domain to the b5 domain. PMID:20630863

  16. Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

    PubMed

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

    2010-09-24

    NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain. PMID:20630863

  17. Congenital Recessive Methemoglobinemia Revealed in Adulthood: Description of a New Mutation in Cytochrome b5 Reductase Gene.

    PubMed

    Forestier, Alexandra; Pissard, Serge; Cretet, Justine; Mambie, Adeline; Pascal, Laurent; Cliquennois, Manuel; Cambier, Nathalie; Rose, Christian

    2015-01-01

    Methemoglobinemia can be acquired (oxidizing drugs or chemicals products) or inherited either by mutations affecting globin chains [M hemoglobins (M Hbs)] or by defects in the enzymatic system involved in the reduction of spontaneous Hb oxidation: nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. It is encoded by the CYB5R3 gene: there are two phenotypes of autosomal recessive congenital methemoglobinemia, in type II CYB5R deficiency is generalized and affects all cells, leading to an early onset, whereas in type I, the enzyme deficiency is restricted to erythrocytes, usually discovered in infancy but not exclusively. We report a new case of methemoglobinemia discovered in a patient from Bahrain who exhibited an unknown dyspnea at the age of 37 years without trigger events or oxidizing products. We discovered a new mutation in the CYB5R3 gene: exon 9, codon 266 (delGAG) (GLU) (CYB5R3: c.726_729delGAG) in the homozygous state. Appearance of methemoglobinemia in an adult usually suggests an acquired cause but our case illustrated that it could also reveal a type I mutation of cytochrome b5 reductase. PMID:26291966

  18. Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.

    PubMed

    Yamazaki, H; Johnson, W W; Ueng, Y F; Shimada, T; Guengerich, F P

    1996-11-01

    Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450. PMID:8910324

  19. A novel L218P mutation in NADH-cytochrome b5 reductase associated with type I recessive congenital methemoglobinemia.

    PubMed

    Arikoglu, Tugba; Yarali, Nese; Kara, Abdurrahman; Bay, Ali; Bozkaya, Ikbal O; Tunc, Bahattin; Percy, Melanie J

    2009-01-01

    The presence of central cyanosis that is unrelated to cardiopulmonary causes alerts clinicians to a possible diagnosis of methemoglobinemia. Congenital methemoglobinemia due to deficiency of nicotinamide-adenine dinucleotide (NADH)-cytochrome b5 reductase (cb(5)r) is an autosomal recessive disorder characterized by life long cyanosis. Here we report a six-year old boy who presented with central cyanosis and upon examination revealed a methemoglobin level of 19.0%. Sequencing the CYB5R3 gene identified a homozygous T-->C transition at base c.653, which changed codon 218 from leucine to proline (L218P) in cb(5)r protein. Treatment with ascorbic acid relieved the cyanosis and returned methemoglobin levels to normal. PMID:19579085

  20. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  1. Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria*

    PubMed Central

    Neve, Etienne P. A.; Nordling, Åsa; Andersson, Tommy B.; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-01-01

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b5 reductase (CYB5R), cytochrome b5 (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b5 type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b5 type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  2. Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

    PubMed

    Neve, Etienne P A; Nordling, Asa; Andersson, Tommy B; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-02-24

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  3. The N-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

    PubMed Central

    Jakobs, Heyka H.; Mikula, Michal; Havemeyer, Antje; Strzalkowska, Adriana; Borowa-Chmielak, Monika; Dzwonek, Artur; Gajewska, Marta; Hennig, Ewa E.; Ostrowski, Jerzy; Clement, Bernd

    2014-01-01

    The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism. PMID:25144769

  4. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

    PubMed

    Kugler, W; Pekrun, A; Laspe, P; Erdlenbruch, B; Lakomek, M

    2001-04-01

    Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. PMID:11295830

  5. A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase

    PubMed Central

    1992-01-01

    Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid- specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms. PMID:1577871

  6. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    PubMed

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  7. The induction of microsomal NADPH:cytochrome P450 and NADH:cytochrome b(5) reductases by long-term salt treatment of cotton (Gossypium hirsutum L.) and bean (Phaseolus vulgaris L.) plants.

    PubMed

    Brankova, Liliana; Ivanov, Sergei; Alexieva, Vera

    2007-09-01

    We studied the effect of salinity on the activity of microsomal NADPH:cytochrome P450 reductase (CPR, EC 1.6.2.4) and NADH:ferricytochrome b(5) oxidoreductase (B5R, EC 1.6.2.2) in two dicotyledonous plant species differing in their sensitivity to salt, cotton (Gossypium hirsutum L. cv Ogosta) and common bean (Phaseolus vulgaris L. cv Dobrujanski 7). A significant inhibition of fresh weight of salt-treated bean plants was observed, while cotton was affected to a much lesser degree. NaCl application resulted in a significant increase in the activity of both reductases, but was more pronounced in salt-tolerant cotton. We suppose that alterations in B5R and CPR activities may be targeted to the maintenance of membrane lipids. Most probably, plants use both enzymes (B5R and CPR) and their respective electron donors (NADH and NADPH) to reduce cytochrome b(5), which can donate reducing equivalents to a series of lipid-modification reactions such as desaturation and hydroxylation.

  8. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability*

    PubMed Central

    Rahaman, Md. Mizanur; Reinders, Fabio G.; Koes, David; Nguyen, Anh T.; Mutchler, Stephanie M.; Sparacino-Watkins, Courtney; Alvarez, Roger A.; Miller, Megan P.; Cheng, Dongmei; Chen, Bill B.; Jackson, Edwin K.; Camacho, Carlos J.; Straub, Adam C.

    2015-01-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μm) and ZINC39395747 (IC50 = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  9. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability.

    PubMed

    Rahaman, Md Mizanur; Reinders, Fabio G; Koes, David; Nguyen, Anh T; Mutchler, Stephanie M; Sparacino-Watkins, Courtney; Alvarez, Roger A; Miller, Megan P; Cheng, Dongmei; Chen, Bill B; Jackson, Edwin K; Camacho, Carlos J; Straub, Adam C

    2015-07-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μM) and ZINC39395747 (IC50 = 9.14 μM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  10. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A.

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  11. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence

    PubMed Central

    Derbyshire, Mark C.; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J.; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-01-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC–MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  12. NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

    PubMed

    Lund, Rikke R; Leth-Larsen, Rikke; Caterino, Tina Di; Terp, Mikkel G; Nissen, Jeanette; Lænkholm, Anne-Vibeke; Jensen, Ole N; Ditzel, Henrik J

    2015-11-01

    Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are

  13. The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities.

    PubMed

    Billen, M J; Squires, E J

    2009-01-01

    Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5alpha-androst-16-en-3-one (5alpha-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17alpha-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-beta synthase as well as the 17alpha-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17alpha-hydroxylase (p<0.013), a transient increase in C17,20 lyase, and an increase in andien-beta synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17alpha-hydroxylase, but did not affect the andien-beta synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1. PMID:19101629

  14. Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.

    PubMed Central

    Chester, D W; Skita, V; Young, H S; Mavromoustakos, T; Strittmatter, P

    1992-01-01

    Cytochrome b5 is a microsomal membrane protein which provides reducing potential to delta 5-, delta 6-, and delta 9-fatty acid desaturases through its interaction with cytochrome b5 reductase. Low angle x-ray diffraction has been used to determine the structure of an asymmetrically reconstituted cytochrome b5:DMPC model membrane system. Differential scanning calorimetry and fluorescence anisotropy studies were performed to examine the bilayer physical dynamics of this reconstituted system. These latter studies allow us to constrain structural models to those which are consistent with physical dynamics data. Additionally, because the nonpolar peptide secondary structure remains unclear, we tested the sensitivity of our model to different nonpolar peptide domain configurations. In this modeling approach, the nonpolar peptide moiety was arranged in the membrane to meet such chemically determined criteria as protease susceptibility of carboxyl- and amino-termini, tyrosine availability for pH titration and tryptophan 109 location, et cetera. In these studies, we have obtained a reconstituted cytochrome b5:DMPC bilayer structure at approximately 6.3 A resolution and conclude that the nonpolar peptide does not penetrate beyond the bilayer midplane. Structural correlations with calorimetry, fluorescence anisotropy and acyl chain packing data suggest that asymmetric cytochrome b5 incorporation into the bilayer increases acyl chain order. Additionally, we suggest that the heme peptide:bilayer interaction facilitates a discreet heme peptide orientation which would be dependent upon phospholipid headgroup composition. Images FIGURE 1 FIGURE 2 FIGURE 7 PMID:1600082

  15. Polymer phase partition in the purification of cytochrome P-450 and cytochrome b5 from the yeast Brettanomyces anomalus.

    PubMed

    Kärenlampi, S O; Nikkilä, H; Hynninen, P H

    1986-02-01

    About 0.5% of the total cellular protein in the yeast Brettanomyces anomalus is membrane-bound cytochrome P-450, when this yeast is grown in the presence of 5% glucose as the main carbon and energy source. A partial purification of cytochrome P-450 by phase partition is described. Breakdown of yeast cell walls with microbial enzyme preparations led to extensive losses of this hemoprotein. Instead, by a carefully controlled mechanical breakage as much as 50% of the total cellular cytochrome P-450 could be recovered. During the solubilization of cytochrome P-450 from the cell homogenate with Triton X-100, the protective agents dithiothreitol, EDTA, and butylated hydroxytoluene prevented major losses of the hemoprotein. Applying a three-phase partition system (polyethylene glycol-Ficoll-dextran) to the solubilized whole cell homogenate in the presence of 1 M sodium chloride, followed by a precipitation of the top "oily layer" with 25% polyethylene glycol, a 25- to 60-fold enrichment of cytochrome P-450 was obtained. This corresponds to a specific content of 0.8-2.2 nmol of cytochrome P-450 per milligram of protein. Cytochrome b5 enriched (41%) to the PEG-Ficoll interphase, and NADPH-cytochrome c reductase and "cytochromes P-420" to the Ficoll and dextran phases. The polymer phase partition system thus serves as an excellent initial purification step of cytochrome P-450 without a need for the preparation of the microsomal fraction. Another advantage of the method is that it allows the simultaneous partial purification of cytochrome b5. PMID:3828082

  16. Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

    PubMed Central

    Qian, Chengmin; Yao, Yong; Ye, Keqiong; Wang, Jinfeng; Tang, Wenxia; Wang, Yunhua; Wang, Wenhu; Lu, Junxia; Xie, Yi; Huang, Zhongxian

    2001-01-01

    The solution structure of oxidized bovine microsomal cytochrome b5 mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045±0.009 nm and 0.088±0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b5. The binding of different surface mutants of cytochrome b5 with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b5 surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b5, do occur in the association between cytochrome b5 and cytochrome c. PMID:11714912

  17. The role of cytochrome b5 structural domains in interaction with cytochromes P450.

    PubMed

    Sergeev, G V; Gilep, A A; Usanov, S A

    2014-05-01

    To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.

  18. Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b₅ in parasitic adaptation.

    PubMed

    Takamiya, Shinzaburo; Hashimoto, Muneaki; Mita, Toshihiro; Yokota, Takehiro; Nakajima, Yoshitaka; Yamakura, Fumiyuki; Sugio, Shigetoshi; Fujimura, Tsutomu; Ueno, Takashi; Yamasaki, Hiroshi

    2016-04-01

    We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

  19. A spectroscopic study of uranyl-cytochrome b5/cytochrome c interactions

    NASA Astrophysics Data System (ADS)

    Sun, Mei-Hui; Liu, Shuang-Quan; Du, Ke-Jie; Nie, Chang-Ming; Lin, Ying-Wu

    2014-01-01

    Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO22+) to interact with various proteins and disturb their biological functions. Cytochrome b5 (cyt b5) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b5-cyt c complex. In previous molecular modeling study in combination with UV-Vis studies, we found that UO22+ is capable of binding to cyt b5 at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b5 and cyt c, as well as cyt b5-cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b5 (KD = 10 μM), cyt c (KD = 87 μM), and cyt b5-cyt c complex (KD = 30 μM) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b5-cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b5/cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity.

  20. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  1. The cytochrome bd respiratory oxygen reductases.

    PubMed

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.

  2. Giardia intestinalis Incorporates Heme into Cytosolic Cytochrome b5

    PubMed Central

    Pyrih, Jan; Harant, Karel; Martincová, Eva; Sutak, Robert; Lesuisse, Emmanuel; Hrdý, Ivan

    2014-01-01

    The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated. PMID:24297440

  3. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  4. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

  5. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Iablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Moskaleva, N E; Bulko, T V; Shumiantseva, V V; Archakov, A I

    2014-01-01

    Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

  6. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Yablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Bulko, T V; Moskaleva, N E; Shumyantseva, V V; Archakov, A I

    2015-01-01

    Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

  7. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides.

  8. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells

    PubMed Central

    Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.

    2016-01-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  9. Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching.

    PubMed

    Markello, T; Zlotnick, A; Everett, J; Tennyson, J; Holloway, P W

    1985-06-01

    Cytochrome b5, a protein isolated from the endoplasmic reticulum by detergent extraction, interacts spontaneously with small unilamellar phosphatidylcholine vesicles. When the vesicles are made from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), the tryptophan fluorescence of the cytochrome is enhanced, and when they are made from 1-palmitoyl-2-(dibromostearoyl) phosphatidylcholine (BRPC), the fluorescence is quenched. A series of BRPC were synthesized with bromine atoms at the 6,7, 9,10, 11,12 or 15,16 positions. The vesicles synthesized from each of these lipids were similar in size to those made from POPC. The relative fluorescence intensities of the cytochrome b5 in POPC and 6,7-, 9,10-, 11,12- and 15,16- BRPC were 100, 19.4, 29.4, 37.1, and 54.0, respectively. These data suggest that the exposed tryptophan(s) is (are) at a depth of 0.7 nm below the surface of the vesicle. Bromine is a collisional quencher; hence, these data may indicate the relative position of the lipid annulus around the protein rather than the depth of the protein below the average vesicle surface. Cytochrome b5 contains three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three are fluorescent with an average quantum yield, when in POPC vesicles, of 0.21. Fluorescence lifetime measurements by the demodulation technique indicated heterogeneity of fluorescence lifetimes in all vesicles. The lifetimes in the BRPC vesicles ranged from 2.0 to 2.4 ns compared to a value of 3.3 ns in POPC.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  11. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  12. Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

    DOEpatents

    Craft, David L.; Madduri, Krishna M.; Loper, John C.

    2003-01-01

    A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

  13. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  14. Purification and characterization of two forms of cytochrome b5 from an arachidonic acid-producing fungus, Mortierella hygrophila.

    PubMed

    Kouzaki, N; Kawashima, H; Chung, M C; Shimizu, S

    1995-06-01

    Two forms of cytochrome b5 have been purified from the microsomes of an arachidonic acid-producing fungus, Mortierella hygrophila IFO 5941, after detergent solubilization. They have monomeric molecular masses of about 16 kDa and 19 kDa. Their absorption spectra are similar to those of mammalian cytochrome b5s. Their amino acid compositions show some similarity to those of mammalian cytochrome b5s, but the contents of some amino acids (glycine, alanine, aspartic acid + asparagine, glutamic acid + glutamine, arginine, proline, histidine, leucine and lysine) are unique to the cytochrome b5s of M. hygrophila. Some of their internal peptide sequences also show close homology with those of some mammals (approx. 65 to 67%), while some others show no or little homology. The addition of various acyl-CoAs to NADH-reduced microsomes caused an abrupt shiftdown of the steady state reduction level of cytochrome b5. This indicates the increased utilization of electrons for the desaturation process and may suggest that the cytochrome b5s of this fungus actually take part in its microsomal desaturation system for polyunsaturated fatty acid biosynthesis as electron carriers. PMID:7786894

  15. Participation of NADPH-cytochrome C reductase in thyroid hormone biosynthesis.

    PubMed

    Yamamoto, K; DeGroot, L J

    1975-04-01

    Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid. PMID:235416

  16. Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.

    PubMed

    Kumar, Santosh; Davydov, Dmitri R; Halpert, James R

    2005-08-01

    The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool. PMID:15870379

  17. Separation of microsomal cytochrome b5 via phase separation in a mixed solution of Triton X-114 and charged dextran.

    PubMed

    Tani, H; Ooura, T; Kamidate, T; Kamataki, T; Watanabe, H

    1998-04-24

    The successful introduction of a charged dextran into the Triton X-114 phase separation system for the selective extraction of cytochrome b5 (cyt. b5) in liver microsomes is described. In the absence of charged dextran, 55% of total microsomal proteins and 84% of cyt. b5 were extracted into the surfactant-rich phase. In the presence of anionic dextran sulfate, the extractability of total microsomal proteins was greatly reduced while that of cyt. b5 was increased. After triplicate extraction, cyt. b5 was purified more than 10-fold from microsomes with a recovery of 91% in the surfactant-rich phase. In view of its operational simplicity, this method provides a good means for the partial purification of cyt. b5 prior to chromatographic separations.

  18. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  19. The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells*

    PubMed Central

    Plitzko, Birte; Ott, Gudrun; Reichmann, Debora; Henderson, Colin J.; Wolf, C. Roland; Mendel, Ralf; Bittner, Florian; Clement, Bernd; Havemeyer, Antje

    2013-01-01

    The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system. PMID:23703616

  20. Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.

    PubMed

    Hatakeyama, Mayumi; Kitaoka, Takuya; Ichinose, Hirofumi

    2016-07-01

    Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.

  1. Studies on NADH (NADPH)-cytochrome c reductase (FMN-containing) from yeast. Isolation and physicochemical properties of the enzyme from top-fermenting ale yeast.

    PubMed

    Johnson, M S; Kuby, S A

    1985-10-01

    Only three major NADPH-nitrotetrazolium blue (NTB) reductases may be detected in a unique top-ale yeast (Saccharomyces cerevisiae, Narragansett strain), which appears to be of a near anaerobic type with the absence of cytochromes c and a/a3 and the presence of cytochromes P-450 and b5. Two of these three major NADPH-NTB reductases possessed NADH-NTB reductase activity; the third was specific for NADPH and was isolated in this laboratory (Tryon, E., Cress, M. C., Hamada, M., and Kuby, S. A. (1979) Arch. Biochem. Biophys. 197, 104-118) vis. NADPH-cytochrome c reductase (FAD-containing). A description of the isolation procedure is provided for one of these two NADH(NADPH)-NTB reductases, viz. NADH(NADPH)-cytochrome c reductase (FMN-containing), which accounts for about one-half of the total cyanide-insensitive menadione-activated respiration of this yeast. This NADH(NADPH)-cytochrome c reductase has been isolated from an extract of an acetone powder of the top-fermenting ale yeast, with an apparent purification of more than 67-fold and a final specific activity of 0.41 and 0.31 mumol/min/mg for NADH- and NADPH-dependent reduction, respectively. The isolated enzyme proved to be homogeneous by electrophoresis on cellulose acetate and on polyacrylamide gels. It had a pI of 5.25 (at gamma/2 = 0.05) and a molecular size under nondenaturing conditions (as determined by chromatography on Sephadex G-100 and Sephacryl S-200) of 70,000 daltons. On denaturation, the enzyme dissociated into two similar, if not identical, subunits which possessed a molecular weight of 34,000 by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis and a weight average molecular weight of 35,000 by sedimentation equilibrium in the presence of 4.0 M guanidinium chloride. The absorbance spectrum of NADH(NADPH)-cytochrome c reductase (FMN-containing) showed three maxima at 464, 383, and 278 nm, with extinction coefficients of 9.88, 9.98, and 64.6 mM-1 cm-1, respectively. The reductase, as

  2. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  3. Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

    PubMed

    Jeřábek, Petr; Florián, Jan; Martínek, Václav

    2016-04-18

    Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex. PMID:26918755

  4. Evolving the [Myoglobin, Cytochrome b5] Complex from Dynamic Toward Simple Docking: Charging the Electron-Transfer Reactive Patch

    PubMed Central

    Trana, Ethan N.; Nocek, Judith M.; Knutson, Amanda K.; Hoffman, Brian M.

    2012-01-01

    We describe photo-initiated electron transfer (ET) from a suite of Zn-substituted myoglobin (1Mb) variants to cytochrome b5 (b5). An electrostatic interface redesign strategy has led to the introduction of positive charges in the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at `hotspot' residues (D44, D60, E85), augmented by the elimination of negative charges from Mb or b5 by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges: −5 < −qMbqb5 < 40. The binding affinity (Ka) increases a thousand-fold as −qMbqb5 increases through this range, and exhibits a surprisingly simple, exponential dependence on −qMbqb5. This is explained in terms of electrostatic interactions between a `charged reactive patch' (crp) on each partner's surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant, and (ii) encompasses a major fraction of the `reactive region' (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As −qMbqb5 increases, the complex undergoes a transition from fast to slow exchange dynamics on the triplet ET timescale, with a correlated progression in the rate constants for intracomplex (ket) and bimolecular (k2) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET, and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by applying it to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c2. Deviations from this model also are discussed. PMID:23067206

  5. Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5.

    PubMed

    Stiborová, Marie; Poljaková, Jitka; Martínková, Eva; Ulrichová, Jitka; Simánek, Vilím; Dvořák, Zdeněk; Frei, Eva

    2012-12-16

    Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine-DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b(5) on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine-DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b(5) increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N(2)-oxide. CYP3A4 and 1A1 in the presence of cytochrome b(5) are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine-DNA adduct 2. These ellipticine-DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC(50) value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

  6. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

  7. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  8. Cytochrome b5 null mouse: a new model for studying inherited skin disorders and the role of unsaturated fatty acids in normal homeostasis.

    PubMed

    Finn, Robert D; McLaughlin, Lesley A; Hughes, Catherine; Song, Chengli; Henderson, Colin J; Roland Wolf, C

    2011-06-01

    Microsomal cytochrome b (5) is a ubiquitous, 15.2 kDa haemoprotein implicated in a number of cellular processes such as fatty acid desaturation, drug metabolism, steroid hormone biosynthesis and methaemoglobin reduction. As a consequence of these functions this protein has been considered essential for life. Most of the ascribed functions of cytochrome b (5), however, stem from in vitro studies and for this reason we have carried out a germline deletion of this enzyme. We have unexpectedly found that cytochrome b (5) null mice were viable and fertile, with pups being born at expected Mendelian ratios. However, a number of intriguing phenotypes were identified, including altered drug metabolism, methaemoglobinemia and disrupted steroid hormone homeostasis. In addition to these previously identified roles for this protein, cytochrome b (5) null mice displayed skin defects closely resembling those observed in autosomal recessive congenital ichthyosis and retardation of neonatal development, indicating that this protein, possibly as a consequence of its role in the de novo biosynthesis of unsaturated fatty acids, plays a central role in skin development and neonatal nutrition. Results from fatty acid profile analysis of several tissues suggest that cytochrome b (5) plays a role controlling saturated/unsaturated homeostasis. These data demonstrate that regional concentrations of unsaturated fatty acids are controlled by endogenous metabolic pathways and not by diet alone.

  9. Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast.

    PubMed

    Johnson, M S; Kuby, S A

    1986-02-15

    A study of the steady-state kinetics of NADH(NADPH)-cytochrome c reductase (FMN-containing) from ale yeast (M. S. Johnson and S. A. Kuby (1985) J. Biol. Chem. 260, 12341-12350) has led to a postulated three-substrate random-ordered hybrid mechanism, where NAD(P)H and FMN add randomly and very likely in a steady-state fashion, followed by an ordered addition of cytochrome c. Kinetic parameters have been derived from this mechanism. Arrhenius plots showed large differences between NADH and NADPH, as the substrate-reductant. Menadione accelerated cytochrome c reduction and also O2 uptake, but vitamin K1 and coenzyme Q10 were ineffective as electron mediators, possibly as a result of their insolubility. With NADPH as the substrate-reductant, the order of the rate of reduction of electron acceptors was ferricyanide greater than DCIP greater than cytochrome c greater than oxygen; with menadione, the specificity sequence was cytochrome c greater than ferricyanide greater than DCIP greater than oxygen. With NADH, the order was ferricyanide greater than cytochrome c greater than oxygen greater than DCIP, which changed to cytochrome c greater than ferricyanide greater than oxygen greater than DCIP on addition of menadione. Cytochrome b5 was also reduced in the absence of oxygen. No transhydrogenase activity was observed, but the reduced thionicotinamide analogs of NADH and NADPH acted as substrates. Superoxide dismutase inhibited cytochrome c reduction in air by 50%, but O2-. was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O2. The product of the reaction with oxygen appeared to be H2O2.

  10. Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis

    PubMed Central

    Kratzer, Regina; Leitgeb, Stefan; Wilson, David K.; Nidetzky, Bernd

    2005-01-01

    Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo–keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/Km) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 Å (1 Å=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8–9 kJ/mol. PMID:16336198

  11. Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy.

    PubMed

    Kotrbová, Věra; Mrázová, Barbora; Moserová, Michaela; Martínek, Václav; Hodek, Petr; Hudeček, Jiří; Frei, Eva; Stiborová, Marie

    2011-09-15

    Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine-DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b(5) enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine-DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b(5) might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b(5) in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b(5) and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.

  12. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

    NASA Astrophysics Data System (ADS)

    Bredt, David S.; Hwang, Paul M.; Glatt, Charles E.; Lowenstein, Charles; Reed, Randall R.; Snyder, Solomon H.

    1991-06-01

    Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.

  13. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence for interactions across the dimeric interface

    PubMed Central

    Judd, Evan T.; Youngblut, Matthew; Pacheco, A. Andrew; Elliott, Sean J.

    2013-01-01

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer. PMID:23210513

  14. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface.

    PubMed

    Judd, Evan T; Youngblut, Matthew; Pacheco, A Andrew; Elliott, Sean J

    2012-12-21

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

  15. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

    PubMed

    Shao, Fang-Yuan; Du, Zhi-Yun; Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-Dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-10-13

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy. PMID:26439985

  16. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

    PubMed

    Shao, Fang-Yuan; Du, Zhi-Yun; Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-Dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-10-13

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

  17. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  18. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  19. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  20. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. PMID:20561902

  1. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Mancinelli, R. L.; Cronin, S.; Hochstein, L. I.

    1986-01-01

    Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

  2. Electrostatic potentials and electrostatic interaction energies of rat cytochrome b5 and a simulated anion-exchange adsorbent surface.

    PubMed Central

    Roush, D J; Gill, D S; Willson, R C

    1994-01-01

    Electrostatic potentials were determined for the soluble tryptic core of rat cytochrome b5 (using a structure derived from homology modeling) and a simulated anion-exchange surface through application of the linearized finite-difference Poisson-Boltzmann equation with the simulation code UHBD. Objectives of this work included determination of the contributions of the various charged groups on the protein surface to electrostatic interactions with a simulated anion-exchange surface as a function of orientation, separation distance, and ionic strength, as well as examining the potential existence of a preferred contact orientation. Electrostatic interaction free energies for the complex of the model protein and the simulated surface were computed using the electrostatics section of UHBD employing a 110(3) grid. An initial coarse grid spacing of 2.0 A was required to obtain correct boundary conditions. The boundary conditions of the coarse grid were used in subsequent focusing steps until the electrostatic interaction free energies were relatively independent of grid spacing (at approximately 0.5 A). Explicit error analyses were performed to determine the effects of grid spacing and other model assumptions on the electrostatic interaction free energies. The computational results reveal the presence of a preferred interaction orientation; the interaction energy between these two entities, of opposite net charge, is repulsive over a range of orientations. The electrostatic interaction free energies appear to be the summation of multiple fractional interactions between the protein and the anion-exchange surface. The simulation results are compared with those of ion-exchange adsorption experiments with site-directed mutants of the recombinant protein. Comparisons of the results from the computational and experimental studies should lead to a better understanding of electrostatic interactions of proteins and charged surfaces. Images FIGURE 4 FIGURE 5 FIGURE 6 PMID:8061185

  3. Genomic and bioinformatic analysis of NADPH-cytochrome P450 reductase in Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Suwanchaichinda, C; Brattsten, L B

    2014-01-01

    The cytochrome P450 monooxygenase (P450) enzyme system is a major mechanism of xenobiotic biotransformation. The nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is required for transfer of electrons from NADPH to P450. One CPR gene was identified in the genome of the malaria-transmitting mosquito Anopheles stephensi Liston (Diptera: Culicidae). The gene encodes a polypeptide containing highly conserved flavin mononucleotide-, flavin adenine dinucleotide-, and NADPH-binding domains, a unique characteristic of the reductase. Phylogenetic analysis revealed that the A. stephensi and other known mosquito CPRs belong to a monophyletic group distinctly separated from other insects in the same order, Diptera. Amino acid residues of CPRs involved in binding of P450 and cytochrome c are conserved between A. stephensi and the Norway rat Rattus norvegicus Berkenhout (Rodentia: Muridae). However, gene structure particularly within the coding region is evidently different between the two organisms. Such difference might arise during the evolution process as also seen in the difference of P450 families and isoforms found in these organisms. CPR in the mosquito A. stephensi is expected to be active and serve as an essential component of the P450 system.

  4. Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex.

    PubMed

    Lockwood, Colin; Butt, Julea N; Clarke, Thomas A; Richardson, David J

    2011-01-01

    The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

  5. Identification of a cytochrome b5-fusion desaturase responsible for the synthesis of triunsaturated sphingolipid long chain bases in the marine diatom Thalassiosira pseudonana.

    PubMed

    Michaelson, Louise V; Markham, Jonathan E; Zäeuner, Simone; Matsumoto, Midori; Chen, Ming; Cahoon, Edgar B; Napier, Johnathan A

    2013-06-01

    Triunsaturated sphingolipid long chain bases (LCBs) have previously been reported in some specialised tissues of marine invertebrates. We report the presence of similar LCBs in the marine diatom Thalassiosira pseudonana and identify the cytochrome b5-fusion desaturase responsible for the introduction of the third double bond at the Δ10 position in d18:3Δ4,8,10. This study extends the catalytic repertoire of the cytochrome b5 fusion desaturase family, also indicating the presence of orthologues in other marine invertebrates. The function of these polyunsaturated sphingolipid LCBs is currently unknown but it was previously suggested that they play an essential role in primitive animals. The identification of the desaturase responsible for their synthesis paves the way for further studies.

  6. Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars1[W][OA

    PubMed Central

    Fan, Ren-Chun; Peng, Chang-Cao; Xu, Yan-Hong; Wang, Xiao-Fang; Li, Yan; Shang, Yi; Du, Shu-Yuan; Zhao, Rui; Zhang, Xiao-Yan; Zhang, Ling-Yun; Zhang, Da-Peng

    2009-01-01

    Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability. PMID:19502355

  7. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.

  8. Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III)

    PubMed

    Nelson, B D; Mendel-Hartvig, I

    1977-10-17

    Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.

  9. Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

    PubMed

    Trofimov, A A; Polyakov, K M; Lazarenko, V A; Popov, A N; Tikhonova, T V; Tikhonov, A V; Popov, V O

    2015-05-01

    Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

  10. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed

    Fujiwara, T; Fukumori, Y

    1996-04-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  11. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450.

    PubMed

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR undergoes large conformational changes. This mini-review discusses the new evidence provided for such conformational changes involving a combination of a "swinging" and "rotating" model and highlights the molecular mechanisms by which formation of these conformations are controlled and thereby enables CPR to serve as an effective electron transferring "nano-machine".

  12. Glycerol allows low-temperature phase separation of membrane proteins solubilized in Triton X-114: application to the purification of plant cytochromes P-450 and b5.

    PubMed

    Werck-Reichhart, D; Benveniste, I; Teutsch, H; Durst, F; Gabriac, B

    1991-08-15

    The potentiality of the Triton X-114 phase separation technique for the purification of proteins from plant microsomal membranes has been investigated. It was shown that glycerol significantly lowers the cloud point of Triton X-114 solutions in water and of Triton X-114 solubilizates from microsomal membranes. It was also established that solubilized membrane components decrease the temperature of Triton X-114 micellar aggregation. Solubilization of microsomal membrane using detergent to protein ratios lower than 3.5, however, resulted in complete inhibition of detergent phase separation. Phase partitioning of Triton X-114 microsomal solubilizates, performed at low temperature (4 degrees C), in the presence of glycerol, provided a very fast and efficient step for the purification of cytochromes P-450 and b5. Conditions allowing optimal recoveries of these cytochromes have been defined.

  13. A Third Subunit in Ancestral Cytochrome c-Dependent Nitric Oxide Reductases

    PubMed Central

    Bricio, C.; Alvarez, L.; San Martin, M.; Schurig-Briccio, L. A.; Gennis, R. B.

    2014-01-01

    Reduction of NO to N2O by denitrifiying bacteria is catalyzed either by a monomeric quinol-nitric oxide reductase (qNor) or by a heterodimeric cytochrome c-dependent nitric oxide reductase (cNor). In ancient thermophilic bacteria belonging to the Thermales and Aquificales phylogenetic groups, the cluster encoding the cNor includes a small third gene (norH), in addition to those encoding homologues to the subunits of a typical cNor (norC and norB). We show in Thermus thermophilus that the three genes are cotranscribed in a single mRNA from an inducible promoter. The isolation of individual nor mutants and the production in vivo of His-tagged NorH protein followed by immobilized-metal affinity chromatography (IMAC) allowed us to conclude that NorH constitutes a third subunit of the cNor from T. thermophilus, which is involved in denitrification in vivo, likely allowing more efficient electron transport to cNor. PMID:24907324

  14. Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide reductase.

    PubMed

    Pisliakov, Andrei V; Hino, Tomoya; Shiro, Yoshitsugu; Sugita, Yuji

    2012-01-01

    Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3) oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed. PMID:22956904

  15. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval)

    PubMed Central

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it’s much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

  16. Regulation of cytochrome b5 gene transcription by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells.

    PubMed

    Huang, Ningwu; Dardis, Andrea; Miller, Walter L

    2005-08-01

    Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances. PMID:15831526

  17. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOEpatents

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  18. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

    PubMed

    Sayanova, O; Shewry, P R; Napier, J A

    1999-10-01

    Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

  19. Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity1

    PubMed Central

    Sayanova, Olga; Shewry, Peter R.; Napier, Johnathan A.

    1999-01-01

    Unlike most other plant microsomal desaturases, the Δ6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Δ6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ6-fatty acid desaturase resulted in the synthesis and accumulation of Δ6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Δ6-desaturase in transgenic plants failed to produce Δ6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Δ6-fatty acid desaturase is essential for enzymatic activity. PMID:10517856

  20. Definition of cytochrome c binding domains by chemical modification: kinetics of reaction with beef mitochondrial reductase and functional organization of the respiratory chain.

    PubMed

    Speck, S H; Ferguson-Miller, S; Osheroff, N; Margoliash, E

    1979-01-01

    An assay has been developed to study the steady-state kinetics of the reduction of cytochrome c by purified beef heart mitochondrial cytochrome c reductase (cytochrome bc(1) complex, complex III). An analogue of coenzyme Q(2) (2,3-dimethoxy-5-methyl-6-decylhydroquinone) was employed as an antimycin-sensitive reductant. The kinetics of reaction of ten different mono(4-carboxy-2,6-dinitrophenyl) derivatives of horse cytochrome c were determined. The modified proteins showed higher apparent K(m) values than the native protein and greater sensitivity to ionic strength, defining an interaction domain on cytochrome c for purified cytochrome c reductase. This interaction site is located on the front surface of the molecule (which contains the exposed heme edge) and surrounds the point at which the positive end of the dipole axis crosses the surface of the protein. The site is similar to that previously determined for mitochondrial cytochrome c oxidase and yeast cytochrome c peroxidase, suggesting that the primary interaction with redox partners is directed by the dipolar charge distribution on cytochrome c. The extensive overlapping of the interaction domains for the mitochondrial cytochrome c oxidase and reductase indicates that cytochrome c must be mobile in order to transfer electrons between them, depending on their relative positions in the membrane. Whether such mobility is necessary in intact mitochondria depends on whether the interactions with the complete membrane-bound system are the same as with the purified components.

  1. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene. PMID:25504221

  2. A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane.

    PubMed

    Zhang, Zhiqiang; Xia, Siqing; Leonard, Didier; Jaffrezic-Renault, Nicole; Zhang, Jiao; Bessueille, François; Goepfert, Yves; Wang, Xuejiang; Chen, Ling; Zhu, Zhiliang; Zhao, Jianfu; Almeida, M Gabriela; Silveira, Célia M

    2009-02-15

    A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples. PMID:18804367

  3. Identification of ubiquinol cytochrome c reductase hinge (UQCRH) as a potential diagnostic biomarker for lung adenocarcinoma

    PubMed Central

    Gao, Feng; Liu, Qicai; Li, Guoping; Dong, Feng; Qiu, Minglian; Lv, Xiaoting; Zhang, Sheng; Guo, Zheng

    2016-01-01

    Ubiquinol cytochrome c reductase hinge (UQCRH) is a novel protein that localizes in the mitochondrial membrane and induces mitochondrial reactive oxygen species (ROS) generation. It had a high expression rate of 87.10% (108/124) in lung adenocarcinoma. Moreover, serum UQCRH level in patients with lung adenocarcinoma was significantly increased compared with that of pneumonia patients (p < 0.0001) and normal control subjects (p < 0.0001). Receiver operating characteristic curve analysis using an optimal cut-off value of 162.65 pg ml−1 revealed sensitivity and specificity for the diagnosis of lung adenocarcinoma of 88.7% and 85.7%, respectively, with an area under the curve of 0.927 (95% CI: 0.892 to 0.962, p < 0.0001). Serum UQCRH discriminates lung adenocarcinoma patients from the population without cancer with considerable sensitivity and specificity, but it does not distinguish between heavy smokers and lung adenocarcinoma patients. Serum UQCRH could be a potential diagnostic biomarker for lung adenocarcinoma. PMID:27358292

  4. Structure of octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens in a complex with phosphate

    SciTech Connect

    Trofimov, A. A.; Polyakov, K. M.; Boiko, K. M.; Filimonenkov, A. A.; Dorovatovskii, P. V.; Tikhonova, T. V.; Popov, V. O.; Koval'chuk, M. V.

    2010-01-15

    Octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR) catalyzes the reduction of nitrite and hydroxylamine to ammonia. The structures of the free enzyme and of the enzyme in complexes with the substrate (nitrite ion) and the inhibitor (azide ion) have been solved previously. In this study we report the structures of the oxidized complex of TvNiR with phosphate and of this complex reduced by europium(II) chloride (1.8- and 2.0-A resolution, the R factors are 15.9 and 16.7%, respectively) and the structure of the enzyme in the complex with cyanide (1.76-A resolution, the R factor is 16.5%), which was prepared by soaking a crystal of the oxidized phosphate complex of TvNiR. In the active site of the enzyme, the phosphate ion binds to the iron ion of the catalytic heme and to the side chains of the catalytic residues Arg131, Tyr303, and His361. The cyanide ion is coordinated to the heme-iron ion and is hydrogen bonded to the residue His361. In the structure of reduced TvNiR, the phosphate ion is bound in the same manner as in the structure of oxidized TvNiR, and the nine{sub c}oordinated europium ion is located on the surface of one of the crystallographically independent monomers of the enzyme.

  5. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  6. Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

    PubMed

    Lemley, C O; Wilson, M E

    2010-10-01

    Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and

  7. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

  8. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450. PMID:26045289

  9. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450.

  10. Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome cd1 Biosynthesis in Pseudomonas stutzeri

    PubMed Central

    Heikkilä, Mari P.; Honisch, Ulrike; Wunsch, Patrick; Zumft, Walter G.

    2001-01-01

    By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N2O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification. PMID:11160097

  11. Molecular population genetics of the NADPH cytochrome P450 reductase (CPR) gene in Anopheles minimus.

    PubMed

    Srivastava, Hemlata; Huong, Ngo Thi; Arunyawat, Uraiwan; Das, Aparup

    2014-08-01

    Development of insecticide resistance (IR) in mosquito vectors is a primary huddle to malaria control program. Since IR has genetic basis, and genes constantly evolve with response to environment for adaptation to organisms, it is important to know evolutionary pattern of genes conferring IR in malaria vectors. The mosquito Anopheles minimus is a major malaria vector of the Southeast (SE) Asia and India and is susceptible to all insecticides, and thus of interest to know if natural selection has shaped variations in the gene conferring IR. If not, the DNA fragment of such a gene could be used to infer population structure and demography of this species of malaria vector. We have therefore sequenced a ~569 bp DNA segment of the NADPH cytochrome P450 reductase (CPR) gene (widely known to confer IR) in 123 individuals of An. minimus collected in 10 different locations (eight Indian, one Thai and one Vietnamese). Two Indian population samples were completely mono-morphic in the CPR gene. In general, low genetic diversity was found with no evidence of natural selection in this gene. The data were therefore analyzed to infer population structure and demography of this species. The 10 populations could be genetically differentiated into four different groups; the samples from Thailand and Vietnam contained high nucleotide diversity. All the 10 populations conform to demographic equilibrium model with signature of past population expansion in four populations. The results in general indicate that the An. minimus mosquitoes sampled in the two SE Asian localities contain several genetic characteristics of being parts of the ancestral population.

  12. Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

    PubMed

    Remacle, J; Fowler, S; Beaufay, H; Amarcostesec, A; Berthet, J

    1976-11-01

    The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

  13. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation.

  14. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. PMID:27243445

  15. The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

    PubMed Central

    Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

    2012-01-01

    Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

  16. Studies on NADPH-cytochrome c reductase. II. Steady-state kinetic properties of the crystalline enzyme from ale yeast.

    PubMed

    Tryon, E; Kuby, S A

    1984-01-01

    From a study of the steady-state kinetics (at pH 7.6, 30 degrees C) of the reduction of cytochrome c, a 'ping-pong' mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae [1], a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates, NADPH and FAD, followed by reactions of the third substrate, Cyt C3+. On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c3+ as the variable substrate). A noncompetitive type of inhibition was also found for cytochrome c2+ with NADPH as variable substrate, in confirmation of the proposed mechanism. With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c3+, a value for KNADPH could be estimated which agreed with that estimated above, with cytochrome c3+ as the acceptor, again, in confirmation of the postulated mechanism. The reactions with molecular O2 catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its Km for O2 estimated to be about 0.15 mmol/l at pH 7.6, 25 degrees C. The product of the reaction appears to be H2O2, which acts as a noncompetitive inhibitor for NADPH (Ki = 0.5 mmol/l), and tentatively an enzyme ternary complex containing oxygen and FADoh (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing Oo2 (superoxide) and FAD.

  17. Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus.

    PubMed

    Ranghino, G; Scorza, E; Sjögren, T; Williams, P A; Ricci, M; Hajdu, J

    2000-09-12

    The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fülöp, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fülöp, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fülöp, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.

  18. Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c.

    PubMed

    Ascenzi, Paolo; Marino, Maria; Polticelli, Fabio; Santucci, Roberto; Coletta, Massimo

    2014-10-01

    Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO2 (-)-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k on for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M(-1) s(-1) (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10(-6) M, (1.8 ± 0.2) × 10(-6) M, and (1.4 ± 0.2) × 10(-6) M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm. PMID:24969400

  19. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b/sub 5/ reductase

    SciTech Connect

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-06-01

    A cDNA coding for human liver NADH-cytochrome b/sub 5/ reductase was cloned from a human liver cDNA library constructed in phage lambdagt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b/sub 5/ reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb/sub 5/R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb/sub 5/R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  20. A Missense Mutation in the Human Cytochrome b5 Gene causes 46,XY Disorder of Sex Development due to True Isolated 17,20 Lyase Deficiency

    PubMed Central

    Idkowiak, Jan; Randell, Tabitha; Dhir, Vivek; Patel, Pushpa; Shackleton, Cedric H. L.; Taylor, Norman F.; Krone, Nils

    2012-01-01

    Context: Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). Objective: We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. Design: We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. Results: All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. Conclusion: We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations. PMID:22170710

  1. Proton-detected 2D radio frequency driven recoupling solid-state NMR studies on micelle-associated cytochrome-b5

    NASA Astrophysics Data System (ADS)

    Pandey, Manoj Kumar; Vivekanandan, Subramanian; Yamamoto, Kazutoshi; Im, Sangchoul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-05-01

    Solid-state NMR spectroscopy is increasingly used in the high-resolution structural studies of membrane-associated proteins and peptides. Most such studies necessitate isotopically labeled (13C, 15N and 2H) proteins/peptides, which is a limiting factor for some of the exciting membrane-bound proteins and aggregating peptides. In this study, we report the use of a proton-based slow magic angle spinning (MAS) solid-state NMR experiment that exploits the unaveraged 1H-1H dipolar couplings from a membrane-bound protein. We have shown that the difference in the buildup rates of cross-peak intensities against the mixing time - obtained from 2D 1H-1H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) experiments on a 16.7-kDa micelle-associated full-length rabbit cytochrome-b5 (cytb5) - can provide insights into protein dynamics and could be useful to measure 1H-1H dipolar couplings. The experimental buildup curves compare well with theoretical simulations and are used to extract relaxation parameters. Our results show that due to fast exchange of amide protons with water in the soluble heme-containing domain of cyb5, coherent 1H-1H dipolar interactions are averaged out for these protons while alpha and side chain protons show residual dipolar couplings that can be obtained from 1H-1H RFDR experiments. The appearance of resonances with distinct chemical shift values in 1H-1H RFDR spectra enabled the identification of residues (mostly from the transmembrane region) of cytb5 that interact with micelles.

  2. Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners.

    PubMed

    Jang, Hyun-Hee; Jamakhandi, Arvind P; Sullivan, Shane Z; Yun, Chul-Ho; Hollenberg, Paul F; Miller, Grover P

    2010-06-01

    As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners. PMID:20152939

  3. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  4. Molecular dynamics simulations give insight into the conformational change, complex formation, and electron transfer pathway for cytochrome P450 reductase

    PubMed Central

    Sündermann, Axel; Oostenbrink, Chris

    2013-01-01

    Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies. PMID:23832577

  5. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

    PubMed

    Silveira, Célia M; Quintas, Pedro O; Moura, Isabel; Moura, José J G; Hildebrandt, Peter; Almeida, M Gabriela; Todorovic, Smilja

    2015-01-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1.

  6. Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

    SciTech Connect

    Stiborova, Marie Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva

    2008-02-01

    Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by {sup 32}P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

  7. Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing.

    PubMed

    Serra, A S; Jorge, S R; Silveira, C M; Moura, J J G; Jubete, E; Ochoteco, E; Cabañero, G; Grande, H; Almeida, M G

    2011-05-01

    In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd(1) (cyt-cd(1)) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c(552) (cyt-c(552)), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 μM cyt-cd(1)/100 μM cyt-c(552) and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c(552) and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254±2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c(552) and cd(1) is 9.9×10(3)M(-1)s(-1). The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 μM; detection and quantification limits of 7 and 24 μM, respectively; sensitivity of 2.49±0.08 Amol(-1)cm(2) μM(-1). Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

  8. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    PubMed

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-01

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  9. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  10. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  11. Neoplastic lesions of the human liver in relation to the activity of the cytochrome P-450 dependent monooxygenase system.

    PubMed

    Plewka, D; Plewka, A; Nowaczyk, G; Kamiński, M; Rutkowski, T; Ludyga, T; Ziaja, K

    2000-01-01

    We studied the activity of Mixed function oxidase (MFO) in human livers affected by cancer. We determined the content of cytochrome P-450 and b5, as well as the activity of their corresponding reductases, according to generally accepted methods. Liver fragments corresponding with a) healthy tissue, b) tissue at the cancer border and, c) cancerous tissue were collected during surgery from patients with liver cancer. We noted that the developing liver cancer decreased the level of cytochrome P-450, even by a magnitude order. The activity of its corresponding reductase was higher in cancerous than in healthy tissues. Cytochrome b5 behaved in an analogous manner, although the decrease in its content was less significant. NADH-cytochrome b5 reductase activity changes were insignificant.

  12. Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

    PubMed

    Pedroso, Humberto A; Silveira, Célia M; Almeida, Rui M; Almeida, Ana; Besson, Stéphane; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

    2016-09-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552. PMID:27133504

  13. Accommodation of two diatomic molecules in cytochrome bo3: insights into NO reductase activity in terminal oxidases†

    PubMed Central

    Hayashi, Takahiro; Lin, Myat T.; Ganesan, Krithika; Chen, Ying; Fee, James A.; Gennis, Robert B.; Moënne-Loccoz, Pierre

    2009-01-01

    Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N2O. Previously, we characterized the heme a3-NO complex formed in cytochrome ba3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to CuB(I) to form an end-on NO-CuB or side-on copper-nitrosyl complex which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N2O. The EPR and resonance Raman signatures of the heme o3-NO complex are comparable to those of the a3-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o3-NO complex does not produce a CuB-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO demonstrate that an [o3–NO • OC–CuB] tertiary complex can form in bo3 but not in ba3. We assign these differences to a greater iron-copper distance in the reduced form of bo3 compared to that of ba3. Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to CuB is not an essential step of the reaction mechanism. PMID:19187032

  14. Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

    PubMed

    Pedroso, Humberto A; Silveira, Célia M; Almeida, Rui M; Almeida, Ana; Besson, Stéphane; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

    2016-09-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552.

  15. The role of cytochromes p450 and aldo-keto reductases in prognosis of breast carcinoma patients.

    PubMed

    Hlaváč, Viktor; Brynychová, Veronika; Václavíková, Radka; Ehrlichová, Marie; Vrána, David; Pecha, Václav; Trnková, Markéta; Kodet, Roman; Mrhalová, Marcela; Kubáčková, Kateřina; Gatěk, Jiří; Vážan, Petr; Souček, Pavel

    2014-12-01

    Metabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and survival of breast carcinoma patients. Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting. AKR1A1 was significantly overexpressed and AKR1C1-4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro. Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in

  16. Development of a novel class of mitochondrial ubiquinol-cytochrome c reductase binding protein (UQCRB) modulators as promising antiangiogenic leads.

    PubMed

    Jung, Hye Jin; Cho, Misun; Kim, Yonghyo; Han, Gyoonhee; Kwon, Ho Jeong

    2014-10-01

    Recently, we identified a novel therapeutic target and a small molecule for regulating angiogenesis. Our study showed that ubiquinol-cytochrome c reductase binding protein (UQCRB) of the mitochondrial complex III plays a crucial role in hypoxia-induced angiogenesis via mitochondrial reactive oxygen species (ROS) mediated signaling. Herein, we developed new synthetic small molecules that specifically bind to UQCRB and regulate its function. To improve the pharmacological properties of 6-((1-hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-one (HDNT), a small molecule that targets UQCRB, a series of HDNT derivatives were designed and synthesized. Several derivatives showed a significant increase in hypoxia inducible factor 1α (HIF-1α) inhibitory potency compared to HDNT. The compounds bound to UQCRB and suppressed mitochondrial ROS-mediated hypoxic signaling, resulting in potent inhibition of angiogenesis without inducing cytotoxicity. Notably, one of these new derivatives significantly suppressed tumor growth in a mouse xenograft model. Therefore, these mitochondrial UQCRB modulators could be potential leads for the development of novel antiangiogenic agents.

  17. Improved free energy profile for reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR).

    PubMed

    Blomberg, Margareta R A; Siegbahn, Per E M

    2016-07-15

    Quantum chemical calculations play an essential role in the elucidation of reaction mechanisms for redox-active metalloenzymes. For example, the cleavage and the formation of covalent bonds can usually not be described only on the basis of experimental information, but can be followed by the calculations. Conversely, there are properties, like reduction potentials, which cannot be accurately calculated. Therefore, computational and experimental data has to be carefully combined to obtain reliable descriptions of entire catalytic cycles involving electron and proton uptake from donors outside the enzyme. Such a procedure is illustrated here, for the reduction of nitric oxide (NO) to nitrous oxide and water in the membrane enzyme, cytochrome c dependent nitric oxide reductase (cNOR). A surprising experimental observation is that this reaction is nonelectrogenic, which means that no energy is conserved. On the basis of hybrid density functional calculations a free energy profile for the entire catalytic cycle is obtained, which agrees much better with experimental information on the active site reduction potentials than previous ones. Most importantly the energy profile shows that the reduction steps are endergonic and that the entire process is rate-limited by high proton uptake barriers during the reduction steps. This result implies that, if the reaction were electrogenic, it would become too slow when the gradient is present across the membrane. This explains why this enzyme does not conserve any of the free energy released. © 2016 Wiley Periodicals, Inc.

  18. The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

    PubMed

    Ascenzi, Paolo; Sbardella, Diego; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo

    2016-06-01

    Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc. PMID:27010463

  19. Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.

    PubMed

    Kratzer, Regina; Kavanagh, Kathryn L; Wilson, David K; Nidetzky, Bernd

    2004-05-01

    Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data

  20. Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase.

    PubMed

    Grunau, Alex; Paine, Mark J; Ladbury, John E; Gutierrez, Aldo

    2006-02-01

    The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez

  1. Mechanisms of integration of de novo-synthesized polypeptides into membranes: signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b5.

    PubMed

    Anderson, D J; Mostov, K E; Blobel, G

    1983-12-01

    We have investigated the in vitro integration into dog pancreas microsomal membranes of three integral membrane proteins that were synthesized de novo in a wheat germ cell-free translation system: calcium ATPase of rabbit sarcoplasmic reticulum, MP26 of bovine lens fiber plasma membrane, and rat liver cytochrome b5. Biosynthetically these proteins show a common feature in that they are synthesized without a transient NH2-terminal signal sequence. Two of these proteins, ATPase and MP26, were shown to require the recently discovered signal-recognition particle (SRP) [Walter, P. & Blobel, G. (1982) Nature (London) 299, 691-698] for integration. By this criterion, therefore, they each contain at least one uncleaved signal sequence. Surprisingly, however, the uncleaved signal sequence(s) of these two proteins did not induce the characteristic SRP-mediated translation arrest that was previously shown for a cleaved signal sequence. Unlike ATPase and MP26, cytochrome b5 did not require SRP for integration into microsomal membrane. Thus, the distinction between an "insertion" sequence (specifying unassisted and opportunistic integration into any exposed membrane) and a "signal" sequence (directing integration into a specific membrane by a receptor-mediated mechanism) is a valid one. By assaying for SRP dependence, the two mechanisms of integration can now be experimentally distinguished.

  2. Progesterone receptor membrane component 1 inhibits the activity of drug-metabolizing cytochromes P450 and binds to cytochrome P450 reductase.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2011-03-01

    Progesterone receptor membrane component 1 (PGRMC1) has been shown to interact with several cytochromes P450 (P450s) and to activate enzymatic activity of P450s involved in sterol biosynthesis. We analyzed the interactions of PGRMC1 with the drug-metabolizing P450s, CYP2C2, CYP2C8, and CYP3A4, in transfected cells. Based on coimmunoprecipitation assays, PGRMC1 bound efficiently to all three P450s, and binding to the catalytic cytoplasmic domain of CYP2C2 was much more efficient than to a chimera containing only the N-terminal transmembrane domain. Down-regulation of PGRMC1 expression levels in human embryonic kidney 293 and HepG2 cell lines stably expressing PGRMC1-specific small interfering RNA had no effect on the endoplasmic reticulum localization and expression levels of P450s, whereas enzymatic activities of CYP2C2, CYP2C8, and CYP3A4 were slightly higher in PGRMC1-deficient cells. Cotransfection of cells with P450s and PGRMC1 resulted in PGRMC1 concentration-dependent inhibition of the P450 activities, and this inhibition was partially reversed by increased expression of the P450 reductase (CPR). In contrast, CYP51 activity was decreased by down-regulation of PGRMC1 and expression of PGRMC1 in the PGRMC1-deficient cells increased CYP51 activity. In cells cotransfected with CPR and PGRMC1, strong binding of CPR to PGRMC1 was observed; however, in the presence of CYP2C2, interaction of PGRMC1 with CPR was significantly reduced, suggesting that CYP2C2 competes with CPR for binding to PGRMC1. These data show that in contrast to sterol synthesizing P450, PGRMC1 is not required for the activities of several drug-metabolizing P450s, and its overexpression inhibits those P450 activities. Furthermore, PGRMC1 binds to CPR, which may influence P450 activity.

  3. The Anticancer Drug Ellipticine Activated with Cytochrome P450 Mediates DNA Damage Determining Its Pharmacological Efficiencies: Studies with Rats, Hepatic Cytochrome P450 Reductase Null (HRN™) Mice and Pure Enzymes

    PubMed Central

    Stiborová, Marie; Černá, Věra; Moserová, Michaela; Mrízová, Iveta; Arlt, Volker M.; Frei, Eva

    2014-01-01

    Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models. PMID:25547492

  4. Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding. Relaxation kinetic studies with coenzyme analogues.

    PubMed

    Gutierrez, Aldo; Munro, Andrew W; Grunau, Alex; Wolf, C Roland; Scrutton, Nigel S; Roberts, Gordon C K

    2003-06-01

    The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy. Previous studies [Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry (2002) 41, 4626-4637] have shown that the observed rate, 1/tau, of interflavin electron transfer (FADsq - FMNsq-->FADox - FMNhq) in CPR reduced at the two-electron level with NADPH is 55 +/- 2 s-1, whereas with dithionite-reduced enzyme the observed rate is 11 +/- 0.5 s-1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer. In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/tau = 18 +/- 0.7 s-1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2'-phosphate is important for enhancing internal electron transfer. To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2',5'-ADP and 2'-AMP. These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/tau = 11 s-1 to 35 +/- 0.2 s-1 and 32 +/- 0.6 s-1, respectively. Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump. Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2',5'-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments. Addition of 2',5'-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to

  5. Elastic rotation of Escherichia coli F{sub O}F{sub 1} having ε subunit fused with cytochrome b{sub 562} or flavodoxin reductase

    SciTech Connect

    Oka, Hideyuki; Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi; Dunn, Stanley D.; Futai, Masamitsu; Iwamoto-Kihara, Atsuko

    2014-04-18

    Highlights: • Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase was observed using a small bead probe. • Carboxyl-terminus of the ε subunit was fused to cytochrome b{sub 562} or flavodoxin reductase. • The F{sub O}F{sub 1} showed continual rotation with similar rate to the wild-type enzyme. • The intra-molecular rotation is flexible and elastic. - Abstract: Intra-molecular rotation of F{sub O}F{sub 1} ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of F{sub O}F{sub 1} with the ε subunit connected to a globular protein [cytochrome b{sub 562} (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.

  6. Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

    PubMed

    Cheng, Xingguo; Zhang, Youcai; Klaassen, Curtis D

    2014-10-01

    NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

  7. Dynamic docking and electron-transfer between cytochrome b5 and a suite of myoglobin surface-charge mutants. Introduction of a functional-docking algorithm for protein-protein complexes.

    PubMed

    Liang, Zhao-Xun; Kurnikov, Igor V; Nocek, Judith M; Mauk, A Grant; Beratan, David N; Hoffman, Brian M

    2004-03-10

    Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb. For each member of this set, we have measured the bimolecular quenching rate constant (k(2)) for the photoinitiated (3)ZnDMb --> Fe(3+)b(5) ET reaction as a function of ionic strength. We find: (i) a dramatic decoupling of binding and reactivity, in which k(2) varies approximately 10(3)-fold within the suite of Mbs without a significant change in binding affinity; (ii) the ET reaction occurs within the "thermodynamic" or "rapid exchange" limit of the "Dynamic Docking" model, in which a large ensemble of weakly bound protein-protein configurations contribute to binding, but only a few are reactive, as shown by the fact that the zero-ionic-strength bimolecular rate constant varies exponentially with the net charge on Mb; (iii) Brownian dynamic docking profiles allow us to visualize the microscopic basis of dynamic docking. To describe these results we present a new theoretical approach which mathematically combines PATHWAY donor/acceptor coupling calculations with Poisson-Boltzmann-based electrostatics estimates of the docking energetics in a Monte Carlo (MC) sampling framework that is thus specially tailored to the intermolecular ET problem. This procedure is extremely efficient because it targets only the functionally active complex geometries by introducing a "reactivity filter" into the computations themselves

  8. Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

    SciTech Connect

    Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.; Nidetzky, Bernd

    2005-01-11

    Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.

  9. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

    PubMed

    Sayanova, O; Smith, M A; Lapinskas, P; Stobart, A K; Dobson, G; Christie, W W; Shewry, P R; Napier, J A

    1997-04-15

    gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

  10. Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

    NASA Astrophysics Data System (ADS)

    Smith, C. I.; Convery, J. H.; Harrison, P.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2014-08-01

    Changes observed in the reflection anisotropy spectroscopy (RAS) profiles of monolayers of cytochrome P450 reductase adsorbed at Au(110)-electrolyte interfaces at 0.056 V following the addition of nicotinamide adenine dinucleotide phosphate (NADP+) are explained in terms of a simple model as arising from changes in the orientation of an isoalloxazine ring located in the flavin mononucleotide binding domain of the protein. The model also accounts for the changes observed in the RAS as the potential applied to the Au(110) surface is varied and suggests that differences in the dependence of the RAS profile of the adsorbed protein on the potential applied to the electrode in the absence and presence of NADP+ are explicable as arising from a competition between the applied potential acting to reduce the protein and the NADP+ to oxidize it.

  11. Chlorate reductase is cotranscribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans.

    PubMed

    Hellberg Lindqvist, Miriam; Nilsson, Thomas; Sundin, Pontus; Rova, Maria

    2015-03-01

    The chlorate-respiring bacterium Ideonella dechloratans is a facultative anaerobe that can use both oxygen and chlorate as terminal electron acceptors. The genes for the enzymes chlorate reductase (clrABDC) and chlorite dismutase, necessary for chlorate metabolism and probably acquired by lateral gene transfer, are located in a gene cluster that also includes other genes potentially important for chlorate metabolism. Among those are a gene for cytochrome c (cyc) whose gene product may serve as an electron carrier during chlorate reduction, a cofactor biosynthesis gene (mobB) and a predicted transcriptional regulator (arsR). Only chlorate reductase and chlorite dismutase have been shown to be expressed in vivo. Here, we report the in vivo production of a single polycistronic transcript covering eight open reading frames including clrABDC, cyc, mobB and arsR. Transcription levels of the cyc and clrA genes were compared to each other by the use of qRT-PCR in RNA preparations from cells grown under aerobic or chlorate reducing anaerobic conditions. The two genes showed the same mRNA levels under both growth regimes, indicating that no transcription termination occurs between them. Higher transcription levels were observed at growth without external oxygen supply. Implications for electron pathway integration following lateral gene transfer are discussed. PMID:25673284

  12. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  13. Study of the Individual Cytochrome b[subscript 5] and Cytochrome b[subscript 5] Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

    SciTech Connect

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R.; Battaile, Kevin P.; Lovell, Scott; Benson, David R.; Zhu, Hao

    2010-09-22

    NADH cytochrome b{sub 5} oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b{sub 5} (b{sub 5}), CHORD-SGT1 (CS), and cytochrome b{sub 5} reductase (b{sub 5}R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b{sub 5} and b{sub 5}R domains (Ncb5or-b{sub 5} and Ncb5or-b{sub 5}R, respectively) and compared them with human microsomal b{sub 5} (Cyb5A) and b{sub 5}R (Cyb5R3). A 1.25 {angstrom} x-ray crystal structure of Ncb5or-b{sub 5} reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His{sup 89} and His{sup 112}, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b{sub 5} family shown to have such a heme environment. Like other b{sub 5} family members, Ncb5or-b{sub 5} has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b{sub 5} differs from Cyb5A with respect to location of the second heme ligand (His{sup 112}) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b{sub 5}R to Ncb5or-b{sub 5} is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b{sub 5} and b{sub 5}R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b{sub 5}R domains suggest that the CS domain facilitates docking of the b{sub 5} and b{sub 5}R domains. Trp{sup 114} is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b{sub 5}R domain to the b{sub 5} domain.

  14. Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

    PubMed

    Pachecka, Jan; Tomaszewski, Piotr; Kubiak-Tomaszewska, Grazyna

    2008-01-01

    Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd).

  15. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

    PubMed Central

    Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang

    2015-01-01

    Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that

  16. Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2014-12-01

    The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85-93%) and CPR I (79-83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus, the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway.

  17. Molecular Cloning, Expression Pattern and Polymorphisms of NADPH-Cytochrome P450 Reductase in the Bird Cherry-Oat Aphid Rhopalosiphum padi (L.)

    PubMed Central

    Zuo, Yayun; Li, Yuting

    2016-01-01

    NADPH–cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, Rhopalosiphum padi, is unavailable. In the current study, a full-length cDNA (2,476 bp) of CPR (RpCPR) encoding 681 amino acids was cloned from R. padi. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, Acyrthosiphon pisum. The mRNA of RpCPR was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. RpCPR expression could also be induced by low concentrations (LC30) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of RpCPR from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that RpCPR may play an important role in the P450-mediated insecticide resistance of R. padi to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, RpCPRmaintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters. PMID:27124302

  18. Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

    SciTech Connect

    Pandey, Amit V.; Flueck, Christa E.; Mullis, Primus E.

    2010-09-24

    Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

  19. The influence of the structure of the Au(110) surface on the ordering of a monolayer of cytochrome P450 reductase at the Au(110)/phosphate buffer interface

    PubMed Central

    Smith, C. I.; Convery, J. H.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2016-01-01

    The reflection anisotropy spectra (RAS) observed initially from Au(110)/phosphate buffer interfaces at applied potentials of −0.652 and 0.056 V are very similar to the spectra observed from ordered Au(110) (1 × 3) and anion induced (1 × 1) surface structures respectively. These RAS profiles transform to a common profile after cycling the potential between these two values over 72 h indicating the formation of a less ordered surface. The RAS of a monolayer of a P499C variant of the human flavoprotein cytochrome P450 reductase adsorbed at 0.056 V at an ordered Au(110)/phosphate buffer interface is shown to arise from an ordered layer in which the optical dipole transitions are in a plane that is orientated roughly normal to the surface and parallel to either the [11̄0] or [001] axes of the Au(110) surface. The same result was found previously for adsorption of P499C on an ordered interface at −0.652 V. The adsorption of P499C at the disordered surface does not result in the formation of an ordered monolayer confirming that the molecular ordering is strongly influenced by both the local structure and the long range macroscopic order of the Au(110) surface.

  20. Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

    PubMed

    Youngblut, Matthew; Pauly, Daniel J; Stein, Natalia; Walters, Daniel; Conrad, John A; Moran, Graham R; Bennett, Brian; Pacheco, A Andrew

    2014-04-01

    Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

  1. The influence of the structure of the Au(110) surface on the ordering of a monolayer of cytochrome P450 reductase at the Au(110)/phosphate buffer interface

    PubMed Central

    Smith, C. I.; Convery, J. H.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2016-01-01

    The reflection anisotropy spectra (RAS) observed initially from Au(110)/phosphate buffer interfaces at applied potentials of −0.652 and 0.056 V are very similar to the spectra observed from ordered Au(110) (1 × 3) and anion induced (1 × 1) surface structures respectively. These RAS profiles transform to a common profile after cycling the potential between these two values over 72 h indicating the formation of a less ordered surface. The RAS of a monolayer of a P499C variant of the human flavoprotein cytochrome P450 reductase adsorbed at 0.056 V at an ordered Au(110)/phosphate buffer interface is shown to arise from an ordered layer in which the optical dipole transitions are in a plane that is orientated roughly normal to the surface and parallel to either the [11̄0] or [001] axes of the Au(110) surface. The same result was found previously for adsorption of P499C on an ordered interface at −0.652 V. The adsorption of P499C at the disordered surface does not result in the formation of an ordered monolayer confirming that the molecular ordering is strongly influenced by both the local structure and the long range macroscopic order of the Au(110) surface. PMID:27630536

  2. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  3. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  4. Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: studies with the hepatic NADPH:cytochrome P450 reductase null (HRN) mouse model.

    PubMed

    Levová, Katerina; Moserová, Michaela; Kotrbová, Vera; Sulc, Miroslav; Henderson, Colin J; Wolf, C Roland; Phillips, David H; Frei, Eva; Schmeiser, Heinz H; Mares, Jaroslav; Arlt, Volker M; Stiborová, Marie

    2011-05-01

    Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

  5. Bioreductive activation of mitoxantrone by NADPH cytochrome P450 reductase does not change its apoptotic stimuli properties in regard to sensitive and multidrug resistant leukaemia HL60 cells.

    PubMed

    Kostrzewa-Nowak, Dorota; Tarasiuk, Jolanta

    2013-12-01

    The objective of this study was to examine the effect of bioreductive activation of antitumour drug, mitoxantrone (MX), by liver NADPH cytochrome P450 reductase (CPR) on inducing apoptosis of human promyelocytic sensitive leukaemia HL60 cell line and its multidrug resistance (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). It was found that non-activated as well as CPR-activated form of MX used at IC90 were able to influence cell cycle of sensitive HL60 as well as resistant cells and induce apoptosis. Interestingly, it was evidenced that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by both studied forms of MX compared to HL60 and HL60/DOX cells. However, the examined agent did not change the expression of Fas receptors on the surface of HL60 sensitive as well as resistant cells regardless of its form used in the study. Obtained results suggest that CPR-dependent reductive activation of MX does not change its apoptotic stimuli properties in regard to sensitive HL60 and multidrug resistant (HL60/VINC and HL60/DOX) leukaemia cells. Nevertheless, taking into account that side toxic effects observed in course of patient treatment with antitumour drugs are dose-dependent, it seems that the reported increase in antiproliferative activity and ability to induce apoptosis of MX after its reductive activation by exogenous CPR against the MDR cells overexpressing both P-glycoprotein and MRP1 at much more lower concentrations of this drug could be of clinical importance for the treatment of tumours resistant to classical chemotherapy. PMID:24076328

  6. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  7. Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

    PubMed

    Stein, Natalia; Love, Daniel; Judd, Evan T; Elliott, Sean J; Bennett, Brian; Pacheco, A Andrew

    2015-06-23

    The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

  8. Reductive activation of the heme iron-nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study.

    PubMed

    Bykov, Dmytro; Neese, Frank

    2012-06-01

    Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.

  9. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT.

  10. Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes.

    PubMed

    Clement, B; Behrens, D; Möller, W; Cashman, J R

    2000-10-01

    For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system, the benzamidoxime reductase, has been described. To reconstitute the complete activity of the benzamidoxime reductase, the system required cytochrome b(5), NADH-cytochrome b(5)-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic process. It was of interest to study the possibility of benzamidoxime reductase reducing N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall, N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines of methamphetamine, amphetamine, and N-methylamine as model compounds were investigated. Formation of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime reductase to their parent amines with reduction rates of 220.6 nmol min(-1) (mg of protein)(-1) for methamphetamine, 5.25 nmol min(-1) (mg of protein)(-1) for amphetamine, and 153 nmol min(-1) (mg of protein)(-1) for N-methylhydroxylamine. Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydroxylamines.

  11. Cytochromes P450--a family of proteins and scientists-understanding their relationships.

    PubMed

    Sue Masters, Bettie; Marohnic, Christopher C

    2006-01-01

    The unifying thread of this review involves NADPH-cytochrome P450 reductase (CYPOR), the microsomal enzyme responsible for transferring electrons to cytochromes P450, as well as several other monooxygenase systems, a lifelong interest of the corresponding author. The intersection of her research with that of Dr. David Kupfer, their resulting collaboration, and the beginning of a long-standing study of fatty acid- and eicosanoid-metabolizing cytochromes P450 (CYP4A gene subfamily), including the role of cytochrome b5, will be reported. The culmination of this interest now involves purification and characterization of the human mutants of CYPOR that have been implicated in pathologies, such as Antley-Bixler syndrome.

  12. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  13. Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system.

    PubMed

    Kuznetsov, Vadim Yu; Ivanov, Yuri D; Archakov, Alexander I

    2004-08-01

    The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm. PMID:15274134

  14. Role of Protein–Protein Interactions in Cytochrome P450-Mediated Drug Metabolism and Toxicity

    PubMed Central

    2015-01-01

    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes. PMID:25133307

  15. Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.

    PubMed

    McCammon, Karen M; Panda, Satya P; Xia, Chuanwu; Kim, Jung-Ja P; Moutinho, Daniela; Kranendonk, Michel; Auchus, Richard J; Lafer, Eileen M; Ghosh, Debashis; Martasek, Pavel; Kar, Rekha; Masters, Bettie Sue; Roman, Linda J

    2016-09-23

    Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

  16. Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.

    PubMed

    McCammon, Karen M; Panda, Satya P; Xia, Chuanwu; Kim, Jung-Ja P; Moutinho, Daniela; Kranendonk, Michel; Auchus, Richard J; Lafer, Eileen M; Ghosh, Debashis; Martasek, Pavel; Kar, Rekha; Masters, Bettie Sue; Roman, Linda J

    2016-09-23

    Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect. PMID:27496950

  17. Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes.

    PubMed

    Whitelaw, Douglas A; Tonkin, Rochelle; Meints, Carla E; Wolthers, Kirsten R

    2015-10-15

    Herein, we compare the kinetic properties of CPR from Arabidopsis thaliana (ATR2), with CPR from Artemisia annua (aaCPR) and human CPR (hCPR). While all three CPR forms elicit comparable rates for cytochrome c(3+) turnover, NADPH reduction of the FAD cofactor is ∼50-fold faster in aaCPR and ATR2 compared to hCPR, with a kobs of ∼500 s(-1) (6 °C). Stopped-flow analysis of the isolated FAD-domains reveals that NADP(+)-FADH2 charge-transfer complex formation is also significantly faster in the plant enzymes, but the rate of its decay is comparable for all three proteins. In hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled to subsequent FAD to FMN electron transfer, indicating that the former catalytic event is slow relative to the latter. In contrast, interflavin electron transfer is slower than NADPH hydride transfer in aaCPR and ATR2, occurring with an observed rate constant of ∼50 s(-1). Finally, the transfer of electrons from FMN to cytochrome c(3+) is rapid (>10(3) s(-1)) in all three enzymes and does not limit catalytic turnover. In combination, the data reveal differences in rate-determining steps between plant CPR and their mammalian equivalent in mediating the flux of reducing equivalents from NADPH to external electron acceptors. PMID:26361974

  18. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

  19. The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells.

    PubMed

    Kostić, T; Andrić, S; Marić, D; Kovacević, R

    1998-07-01

    Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 microg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was significantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data confirmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition. PMID:9712411

  20. Antioxidant activity of new aramide nanoparticles containing redox-active N-phthaloyl valine moieties in the hepatic cytochrome P450 system in male rats.

    PubMed

    Hassan, Hammed H A M; El-Banna, Sabah G; Elhusseiny, Amel F; Mansour, El-Sayed M E

    2012-07-10

    We report the synthesis of aramide nanoparticles containing a chiral N-phthaloyl valine moiety and their antioxidant activities on hepatic contents of cytochrome P₄₅₀, amidopyrene N-demethylase, aniline-4-hyroxylase and induced the hepatic content of cytochrome b5 and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C-reductase. Polymers were obtained as well-separated spherical nanoparticles while highly aggregated particles via H-bonding organization of the aramide-containing pyridine led to a thin layer formation. The effects of the nanoparticles and CCl₄ on enzyme activities and thiobarbituric acid reactive substances (TBARS) levels of male rat liver were studied. Pretreatments of rats with the polyamides prior to the administration of CCl₄ decreased the hepatic content of the tested enzymes. Doses reduced the toxic effects exerted by (•CCl₃) upon the liver through inhibition of the cytochrome P₄₅₀ system. Inhibition of such metabolizing enzymes could reduce the carcinogenic effects of chemical carcinogens.

  1. Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

    PubMed

    Stringer, Rowan A; Ferreira, Suzie; Rose, Jonathan; Ronseaux, Sebastien

    2016-08-01

    The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor.

  2. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

    PubMed

    Crawford, N M; Smith, M; Bellissimo, D; Davis, R W

    1988-07-01

    The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase. PMID:3393528

  3. Suppressive effect of accumulated aluminum trichloride on the hepatic microsomal cytochrome P450 enzyme system in rats.

    PubMed

    Zhu, Yanzhu; Han, Yanfei; Zhao, Hansong; Li, Jing; Hu, Chongwei; Li, Yanfei; Zhang, Zhigang

    2013-01-01

    Aluminum (Al) is a low toxicological metal and can accumulate in the liver. The hepatic microsomal cytochrome P450 enzyme system (CYPS) plays important role in the transformation of the toxic materials. It is not clear if the CYPS is affected by Al exposure. Thus, the aim of this study is to investigate the effects of aluminum trichloride (AlCl(3)) on CYPS in rats. Forty male Wistar rats (5weeks old) weighing 110-120g were randomly allocated and orally exposed to 0, 64.18, 128.36 and 256.72mg/kg body weight (BW) AlCl(3) in drinking water for 120days. The body weight (BW) of rats, hepatosomatic index (HSI), hepatic Al content, the concentrations of cytochrome P450 (CYP450), cytochrome B5 (B5), microsomal protein and the activities of NADPH-cytochrome c reductase (CR), aminopyrin N-demethylase (AND), erythromycin N-demethylase (ERND) and aniline-4-hydeoxylase (AH) were assessed at the end of the experiment. The results showed that the increase in Al concentration decreased BW, HIS, concentrations of CYP450, B5, microsomal protein and the activity of CR, AND, ERND and AH in hepatic microsomes. The results revealed that exposure to AlCl(3) inhibited the microsomal CYP450 dependent enzyme system of liver. Our findings suggest that long term daily exposure of AlCl(3) exerts the suppressive effects and thus may cause dysfunction of hepatic CYP450 dependent enzyme system of rat.

  4. Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

    PubMed

    Cartron, Michaël L; Roldán, M Dolores; Ferguson, Stuart J; Berks, Ben C; Richardson, David J

    2002-12-01

    NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the

  5. Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

    SciTech Connect

    Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, Jim K.; Bigelow, Diana J.; Squier, Thomas C.

    2011-10-14

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent

  6. Interactions between CYP2E1 and CYP2B4: Effects on Affinity for NADPH-Cytochrome P450 Reductase and Substrate Metabolism

    PubMed Central

    Kenaan, Cesar; Shea, Erin V.; Lin, Hsia-lien; Zhang, Haoming; Pratt-Hyatt, Matthew J.

    2013-01-01

    Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a Ki of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent KM and kcat of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent KM of CYP2B4 for CPR increased up to 23-fold with virtually no change in the kcat for the reaction, however, at higher concentrations of CYP2E1, the apparent KM of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the kcat also decreased by 11-fold. Additionally, CYP2E1 increased the apparent KM of CYP2B4 for BNZ by 8-fold and the apparent KM did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent KM values of CYP2B4 and CYP2E1 for CPR are similar, the apparent KM of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR. PMID:23043184

  7. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

  8. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  9. Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes.

    PubMed

    Meyer, Terry E; Tsapin, Alexandre I; Vandenberghe, Isabel; de Smet, Lina; Frishman, Dmitrij; Nealson, Kenneth H; Cusanovich, Michael A; van Beeumen, Jozef J

    2004-01-01

    Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

  10. EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

    PubMed Central

    Huber, Warren J.; Backes, Wayne L.

    2009-01-01

    Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

  11. Cytochrome P450 (CYP2C9*2,*3) & vitamin-K epoxide reductase complex (VKORC1 -1639G

    PubMed Central

    Kaur, Anupriya; Khan, Farah; Agrawal, Suraksha S.; Kapoor, Aditya; Agarwal, Surendra K.; Phadke, Shubha R.

    2013-01-01

    Background & objectives: Studies have demonstrated the effect of CYP2C9 (cytochrome P450) and VKORC1 (vitamin K epoxide reductase complex) gene polymorphisms on the dose of acenocoumarol. The data from India about these gene polymorphisms and their effects on acenocoumarol dose are scarce. The aim of this study was to determine the occurrence of CYP2C9*2,*3 and VKORC 1 -1639G>A gene polymorphisms and to study their effects on the dose of acenocoumarol required to maintain a target International Normalized Ratio (INR) in patients with mechanical heart valve replacement. Methods: Patients from the anticoagulation clinic of a tertiary care hospital in north India were studied. The anticoagulation profile, INR (International Normalized Ratio) values and administered acenocoumarol dose were obtained from the clinical records of patients. Determination of the CYP2C9*2,*3 and VKORC1 -1639G>A genotypes was done by PCR-RFLP (restriction fragment length polymorphism). Results: A total of 111 patients were studied. The genotype frequencies of CYP2C9 *1/*1,*1/*2,*1/*3 were as 0.883, 0.072, 0.036 and that of VKORC1 -1639G>A for GG, AG, and AA genotypes were 0.883, 0.090, and 0.027, respectively. The percentage of patients carrying any of the variant alleles of CYP2C9 and VKORC1 in heterozygous or homozygous form was 34% among those receiving a low dose of ≤20 mg/wk while it was 13.8 per cent in those receiving >20 mg/wk (P=0.014). A tendency of lower dose requirements was seen among carriers of the studied polymorphisms. There was considerable variability in the dose requirements of patients with and without variant alleles. Interpretation & conclusions: The study findings point towards the role of CYP2C9 and VKORC1 gene polymorphisms in determining the inter-individual dose variability of acenocoumarol in the Indian patients with mechanical heart valve replacement. PMID:23481074

  12. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    PubMed

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

  13. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  14. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

  15. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 2 2011-07-01 2011-07-01 false Voting. 242b.5 Section 242b.5 National Defense... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be... notation voting, by voting on material circulated to Regents individually or serially, or by polling...

  16. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  17. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  18. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where the agency...

  19. 7 CFR 15b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Assurances required. 15b.5 Section 15b.5 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 15b.5 Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit...

  20. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  1. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  2. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  3. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  4. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  5. Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae.

    PubMed

    Braun, Martin; Thöny-Meyer, Linda

    2005-09-01

    Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.

  6. 15 CFR 8b.5 - Assurances required.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 1 2011-01-01 2011-01-01 false Assurances required. 8b.5 Section 8b.5... Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit an assurance, on a form specified by the Secretary, that the program or activity will...

  7. 15 CFR 8b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Assurances required. 8b.5 Section 8b.5... Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit an assurance, on a form specified by the Secretary, that the program or activity will...

  8. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  9. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  10. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  11. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  12. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  13. Cytochrome P450 differences in normal and imposex-affected female whelk Buccinum undatum from the open North Sea.

    PubMed

    Santos, M M; ten Hallers-Tjabbes, C C; Vieira, N; Boon, J P; Porte, C

    2002-01-01

    Normal and imposex-affected female Buccinum undatum were sampled from the open North Sea at three locations, one with low, and two with high shipping densities. Cytochrome P450 components and P450 aromatase activity were determined in the microsomal fractions isolated from pooled digestive gland/gonads. Cytochrome P450 aromatase activity was significantly higher (P < 0.05) in normal females collected in the low shipping density area (1,325 +/- 295 fmol/h/mg protein) than levels from imposex animals from a high shipping density area (620 +/- 287 fmol/h/mg protein). A negative correlation was found between aromatase activity and organotin body burden (r = -0.99). Levels of CYP450, cytochrome b5 and NADPH cytochrome c reductase activity did not show differences among groups. This is the first field evidence of depressed aromatase activity in imposex affected females, although additional research under laboratory controlled conditions is required to fully understand the mechanisms underlying the development of imposex in this species.

  14. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  15. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be necessary for the transaction of business. (b) Unless a written ballot is required by a Regent, no actions taken by the Board need be by written ballot. (c) The Chairman of the Board and of each Committee...

  16. Magnetic Circular Dichroism Studies XXV. A Preliminary Investigation of Microsomal Cytochromes*

    PubMed Central

    Dolinger, Peter M.; Kielczewski, Michael; Trudell, James R.; Barth, Günter; Linder, Robert E.; Bunnenberg, Edward; Djerassi, Carl

    1974-01-01

    The application of magnetic circular dichroism as an optical probe for simultaneous identification and determination of at least two microsomal cytochromes is demonstrated. The assignments of the bands in the spectra of microsomal suspensions are made from the spectra of soluble preparations of cytochrome P-450 obtained from Pseudomonas putida and of cytochrome b5 obtained from rat livers. PMID:4521811

  17. CYP345E2, an antenna-specific cytochrome P450 from the mountain pine beetle, Dendroctonus ponderosae Hopkins, catalyses the oxidation of pine host monoterpene volatiles.

    PubMed

    Keeling, Christopher I; Henderson, Hannah; Li, Maria; Dullat, Harpreet K; Ohnishi, Toshiyuki; Bohlmann, Jörg

    2013-12-01

    The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a significant pest of western North American pine forests. This beetle responds to pheromones and host volatiles in order to mass attack and thus overcome the terpenoid chemical defences of its host. The ability of MPB antennae to rapidly process odorants is necessary to avoid odorant receptor saturation and thus the enzymes responsible for odorant clearance are an important aspect of host colonization. An antenna-specific cytochrome P450, DponCYP345E2, is the most highly expressed transcript in adult MPB antenna. In in vitro assays with recombinant enzyme, DponCYP345E2 used several pine host monoterpenes as substrates, including (+)-(3)-carene, (+)-β-pinene, (-)-β-pinene, (+)-limonene, (-)-limonene, (-)-camphene, (+)-α-pinene, (-)-α-pinene, and terpinolene. The substrates were epoxidized or hydroxylated, depending upon the substrate. To complement DponCYP345E2, we also functionally characterized the NADPH-dependent cytochrome P450 reductase and the cytochrome b5 from MPB. DponCYP345E2 is the first cytochrome P450 to be functionally characterized in insect olfaction and in MPB.

  18. Biochemical responses in mussels Perna perna exposed to diesel B5.

    PubMed

    Nogueira, Lílian; Garcia, Danielly; Trevisan, Rafael; Sanches, Ana Letícia Madeira; da Silva Acosta, Daiane; Dafre, Alcir Luiz; Oliveira, Thiago Yukio Kikuchi; de Almeida, Eduardo Alves

    2015-09-01

    In Brazil B5 blend (5% biodiesel and 95% diesel oil) has been adopted as mandatory fuel since 2010 for automotive vehicles. Since little is known about the effects of B5 exposure can promote on antioxidant system of marine biota this study aimed to assess if B5 can generate modifications in antioxidant parameters of mussels Perna perna. To address this question mussels were exposed to two concentrations of B5 (0.01 mL L(-1) and 0.1 mL L(-1)) for 6h, 12h, 48 h and 168 h. Then the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were evaluated in gills and digestive gland as well as the contents of glutathione (GSH) and lipid peroxidation by measuring the malondialdehyde concentration (MDA). In the gills, GST activity decreased after 48 h and GR after 12h of exposure to B5. In digestive glands, the activities of SOD, GPx and GR were changed due to treatments. GSH concentration increased in digestive gland after 6h and 12h and in gills after 48 h for B5 0.1 mL L(-1) and after 168 h in the digestive gland for B5 0.01 mL L(-1) treatment. No lipid peroxidation was detected. The integrated biomarker response index (IBR) evidenced a B5 effect in the digestive gland after 168 h of exposure. Regarding the experimental conditions and species used in this study, long-term exposure to B5 is apparently more likely to affect the parameters tested in P. perna mussels.

  19. Technical description of Stack 296-B-5

    SciTech Connect

    Ridge, T.M.

    1994-11-15

    Of particular concern to facilities on the Hanford site is Title 40, Code of Federal Regulations, Chapter 40, Part 61, Subpart H, ``National emission Standards for Emissions of Radionuclides Other Than Radon From Department of Energy Facilities.`` Assessments of facility stacks and potential radionuclide emissions determined whether these stacks would be subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. Stack 296-B-5 exhausts 221-BB building which houses tanks containing B Plant steam condensate and B Plant process condensate from the operation of the low-level waste concentrator. The assessment of potential radionuclide emissions from the 296-B-5 stack resulted in an effective dose equivalent to the maximally exposed individual of less than 0.1 millirem per year. Therefore, the stack is not subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. However, the sampling and monitoring system must be in compliance with the Environmental Compliance Manual, WHC-CM-7-5. Currently, 296-B-5 is sampled continuously with a record sampler and continuous air monitor (CAM).

  20. Towards engineering increased pantothenate (vitamin B(5)) levels in plants.

    PubMed

    Chakauya, Ereck; Coxon, Katy M; Wei, Ma; Macdonald, Mary V; Barsby, Tina; Abell, Chris; Smith, Alison G

    2008-11-01

    Pantothenate (vitamin B(5)) is the precursor of the 4'-phosphopantetheine moiety of coenzyme A and acyl-carrier protein. It is made by plants and microorganisms de novo, but is a dietary requirement for animals. The pantothenate biosynthetic pathway is well-established in bacteria, comprising four enzymic reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), L: -aspartate-alpha-decarboxylase (ADC), pantothenate synthetase (PS) and ketopantoate reductase (KPR) encoded by panB, panD, panC and panE genes, respectively. In higher plants, the genes encoding the first (KPHMT) and last (PS) enzymes have been identified and characterised in several plant species. Commercially, pantothenate is chemically synthesised and used in vitamin supplements, feed additives and cosmetics. Biotransformation is an attractive alternative production system that would circumvent the expensive procedures of separating racemic intermediates. We explored the possibility of manipulating pantothenate biosynthesis in plants. Transgenic oilseed rape (Brassica napus) lines were generated in which the E. coli KPHMT and PS genes were expressed under a strong constitutive CaMV35SS promoter. No significant change of pantothenate levels in PS transgenic lines was observed. In contrast plants expressing KPHMT had elevated pantothenate levels in leaves, flowers siliques and seed in the range of 1.5-2.5 fold increase compared to the wild type plant. Seeds contained the highest vitamin content, indicating that they might be the ideal target for production purposes.

  1. Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals.

    PubMed

    Berman, M C; Adnams, C M; Ivanetich, K M; Kench, J E

    1976-07-01

    The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 X 10(-3) +/- 0.19 X 10(-3) S-1 (mean +/- S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 X 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 X 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed. PMID:183743

  2. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  3. Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.

    PubMed Central

    Miles, J S; Munro, A W; Rospendowski, B N; Smith, W E; McKnight, J; Thomson, A J

    1992-01-01

    1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments. Images Fig. 1. PMID:1334408

  4. Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

    PubMed

    Park, Ji Won; Reed, James R; Brignac-Huber, Lauren M; Backes, Wayne L

    2014-12-01

    Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

  5. Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris

    USGS Publications Warehouse

    Lovley, D.R.; Widman, P.K.; Woodward, J.C.; Phillips, E.J.P.

    1993-01-01

    The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium- contaminated waters and waste streams.

  6. Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.

    PubMed

    Lovley, D R; Widman, P K; Woodward, J C; Phillips, E J

    1993-11-01

    The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.

  7. Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

    NASA Technical Reports Server (NTRS)

    Tsapin, A. I.; Vandenberghe, I.; Nealson, K. H.; Scott, J. H.; Meyer, T. E.; Cusanovich, M. A.; Harada, E.; Kaizu, T.; Akutsu, H.; Leys, D.; Van Beeumen, J. J.

    2001-01-01

    Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

  8. Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

    PubMed

    Quinn, G B; Trimboli, A J; Prosser, I M; Barber, M J

    1996-03-01

    was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.

  9. Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin.

    PubMed Central

    Correll, C. C.; Ludwig, M. L.; Bruns, C. M.; Karplus, P. A.

    1993-01-01

    The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel beta-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant

  10. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  11. A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella).

    PubMed

    Niu, Guodong; Rupasinghe, Sanjeewa G; Zangerl, Arthur R; Siegel, Joel P; Schuler, Mary A; Berenbaum, May R

    2011-04-01

    The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b(5) on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.

  12. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  13. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  14. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  15. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2000-04-01

    ... 17 Commodity and Securities Exchanges 3 2000-04-01 2000-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a) Each application for a stay of a trustee's duty...

  16. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2011 CFR

    2005-04-01

    ... 17 Commodity and Securities Exchanges 3 2005-04-01 2005-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  17. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  18. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  19. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  20. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  1. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  2. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 9 2014-07-01 2014-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  3. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 9 2011-07-01 2011-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  4. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 9 2013-07-01 2013-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  5. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 9 2012-07-01 2012-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  6. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  7. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  8. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false How will NIH evaluate applications? 52b.5 Section 52b.5 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating...

  9. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false How will NIH evaluate applications? 52b.5 Section 52b.5 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating...

  10. Musk xylene is a novel specific inducer of cytochrome P-450IA2.

    PubMed

    Iwata, N; Minegishi, K; Suzuki, K; Ohno, Y; Kawanishi, T; Takahashi, A

    1992-04-15

    The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

  11. Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome c3

    SciTech Connect

    Wall, Judy D.

    2003-06-01

    The project, ''Reduction of U(VI) and toxic metals by Desulfovibrio cytochrome c3'', is designed to obtain spectroscopic information for or against a functional interaction of cytochrome c3 and uranium in the whole cells. That is, is the cytochrome c3 the uranium reductase? Our approach has been to start with purified cytochrome and determine any unique spectral disturbances during electron flow to U(VI). Then we will attempt to identify these signals emanating from cells actively reducing uranium. This project is being carried out in collaboration with Dr. William Woodruff at the Los Alamos National Laboratory where the spectral experiments are being carried out.

  12. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c.

  13. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c. PMID:21185447

  14. In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

    PubMed Central

    Ketchum, Paul A.; Sevilla, Cynthia L.

    1973-01-01

    In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)–nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH–nitrate reductase, FADH2–nitrate reductase and reduced methyl viologen (MVH)–nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10−4 M Na2MoO4 and 10−2 M NaNO3 to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase. PMID:4270447

  15. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  16. The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis

    SciTech Connect

    Clarke, Thomas A.; Holley, Tracey; Hartshorne, Robert S.; Fredrickson, Jim K.; Zachara, John M.; Shi, Liang; Richardson, David

    2008-10-01

    The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem ‘modules’ similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV–visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

  17. The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis.

    PubMed

    Clarke, Thomas A; Holley, Tracey; Hartshorne, Robert S; Fredrickson, Jim K; Zachara, John M; Shi, Liang; Richardson, David J

    2008-10-01

    The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem 'modules' similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV-visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

  18. Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats.

    PubMed

    Park, So Young; Kim, Chung Hyeon; Lee, Ji Yoon; Jeon, Jang Su; Kim, Min Ju; Chae, Song Hee; Kim, Hyoung Chin; Oh, Soo Jin; Kim, Sang Kyum

    2016-10-01

    In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1'- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

  19. Cytochrome c4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c1 and c5 promote intracellular infection.

    PubMed

    Yip, Emily S; Burnside, Denise M; Cianciotto, Nicholas P

    2011-03-01

    A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c(1) and cycB mutants lacking cytochrome c(5) had normal siderophore expression, cyc4 mutants defective for cytochrome c(4) completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c(4) in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c(5) in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.

  20. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  1. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  2. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  3. 26 CFR 48.4161(b)-5 - Effective date.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... taxes imposed by section 4161(b) are effective with respect to sales made on and after January 1, 1975. ... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Effective date. 48.4161(b)-5 Section 48.4161(b)-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED)...

  4. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 8 Aliens and Nationality 1 2012-01-01 2012-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  5. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  6. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 8 Aliens and Nationality 1 2014-01-01 2014-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  7. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 8 Aliens and Nationality 1 2013-01-01 2013-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  8. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  9. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR...

  10. Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

    PubMed

    Berks, B C; Richardson, D J; Robinson, C; Reilly, A; Aplin, R T; Ferguson, S J

    1994-02-15

    The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve. PMID:8119278

  11. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    PubMed Central

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  12. Li2AlB5O10.

    PubMed

    He, M; Li, H; Chen, X; Xu, Y; Xu, T

    2001-09-01

    A new compound, dilithium aluminium pentaborate, Li(2)AlB(5)O(10), has been synthesized by solid-state reaction and its structure determined by single-crystal X-ray diffraction. This compound is composed of [B(5)O(10)](5-) groups linked by AlO(4) tetrahedra. The [B(5)O(10)](5-) group consists of two hexagonal B-O rings perpendicular to each other connected by tetracoordinated boron. All the B-O rings in this structure can be divided into two groups, with one group approximately parallel and the other perpendicular to the c axis. PMID:11588352

  13. Solubilization and reconstitution of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca

    SciTech Connect

    Desjardins, A.E.; Matthews, D.E.; VanEtten, H.D.

    1984-07-01

    Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tests was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2',5'-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase. 24 references, 4 figures, 4 tables.

  14. Selective and compartmentalized myelin expression of HspB5.

    PubMed

    Quraishe, S; Wyttenbach, A; Matinyarare, N; Perry, V H; Fern, R; O'Connor, V

    2016-03-01

    In the present study, we reveal myelin-specific expression and targeting of mRNA and biochemical pools of HspB5 in the mouse CNS. Our observations are based on in situ hybridization, electron microscopy and co-localization with 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase (CNPase), reinforcing this myelin-selective expression. HspB5 mRNA might be targeted to these structures based on its presence in discrete clusters resembling RNA granules and the presence of a putative RNA transport signal. Further, sub-cellular fractionation of myelin membranes reveals a distinct sub-compartment-specific association and detergent solubility of HspB5. This is akin to other abundant myelin proteins and is consistent with HspB5's association with cytoskeletal/membrane assemblies. Oligodendrocytes have a pivotal role in supporting axonal function via generating and segregating the ensheathing myelin. This specialization places extreme structural and metabolic demands on this glial cell type. Our observations place HspB5 in oligodendrocytes which may require selective and specific chaperone capabilities to maintain normal function and neuronal support.

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

    PubMed Central

    Ramachandra, M; Seetharam, R; Emptage, M H; Sariaslani, F S

    1991-01-01

    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 Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing 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-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin. Images FIG. 2 PMID:1938912

  16. Simulation of multihaem cytochromes.

    PubMed

    Soares, Cláudio M; Baptista, António M

    2012-03-01

    This article presents an overview of the simulation studies of the behaviour of multihaem cytochromes using theoretical/computational methodologies, with an emphasis on cytochrome c(3). It starts with the first studies using rigid molecules and continuum electrostatic models, where protonation and redox events were treated as independent. The gradual addition of physical details is then described, from the inclusion of proton isomerism, to the proper treatment of the thermodynamics of electron-proton coupling, to the explicit inclusion of the solvent and protein structural reorganization into the models, culminating with the method for molecular dynamics simulations at constant pH and reduction potential, where the solvation, conformational, protonation and redox features are all simulated in a fully integrated and coupled way. We end with a discussion of the strategies used to study the interaction between multihaem cytochromes, taking into account the further coupling effect introduced by the molecular association.

  17. Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

    NASA Technical Reports Server (NTRS)

    Velasco, P. J.; Tischner, R.; Huffaker, R. C.; Whitaker, J. R.

    1989-01-01

    Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

  18. Structural stability of W2B5 under high pressure

    NASA Astrophysics Data System (ADS)

    Kumar, N. R. Sanjay; Shekar, N. V. Chandra; Sahu, P. Ch.

    2015-05-01

    High-pressure structural stability studies have been carried out on tungsten boride W2B5 up to maximum pressure of 36 GPa using a Mao-Bell diamond-anvil cell at beamline BR-12 of the ELETTRA synchrotron facility (λ = 0.68881 Å). The hexagonal phase (S.G:P63/mmc) of W2B5 is stable up to the maximum pressure studied. The bulk modulus is estimated to be ~347 GPa using the Birch-Murnaghan equation of state. The variation of lattice parameters and bond lengths B-B and W-B have been studied and the c-axis is seen to be marginally more compressible than the a-axis.

  19. Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment.

    PubMed

    Liu, Yang-Wei; Kelly, David J

    2015-06-01

    The microaerophilic food-borne pathogen Campylobacter jejuni uses complex cytochrome-rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono-haem c-type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di-haem Cj0037 (CccC) are electron donors to the cb-oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site-directed mutagenesis resulted in an almost complete loss of all other c-type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up-regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L-cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c-type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.

  20. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  1. Certain Metal Ions are Inhibitors of Cytochrome b (6) f Complex 'Rieske' Iron-Sulfur Protein Domain Movements

    SciTech Connect

    Roberts, Arthur G.; Bowman, Michael K.; Kramer, David M.

    2002-03-26

    1Abbreviations: cyt, cytochrome; cyt bL, low potential b cytochrome; cyt bH, high potential b cytochrome; DBMIB, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone; DMSO, dimethylsulfoxide; DNP-INT, 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether; EPR, electron paramagnetic resonance; HEPES, n-(2-hydroxyethyl)piperazine-n'-(2-ethanesulfonic acid); NQNO, 2-nonyl-4-hydroxyquinoline n-oxide; ISP, iron-sulfur protein; MOA, E-b-methoxyacrylate; pmf, proton motive force; PC, plastocyanin; PQ, plastoquinone; PQH2, plastoquinol; PS, photosystem; Qi, quinol reductase; Qo, quinol oxidase; UHDBT, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole.

  2. Modification of the redox state of cytochrome c oxidase of rice due to certain stress treatments.

    PubMed

    Dhage, A R; Desai, B B; Naik, R M; Munjal, S V; Naik, M S

    1992-10-01

    The redox state of cytochrome alpha 3 during in situ respiration of leaves of 20-day-old rice seedlings was assessed by in vivo aerobic assay of nitrate reductase, after 1 min exposure to carbon monoxide. Different stress treatments like water and salt stresses, disintegration of leaf tissues and darkness modified the redox state of cytochrome c oxidase. The dark treatment altered the redox state of cytochrome oxidase from reduced to the oxidized state, as judged by its reaction with CO in CO-sensitive rice cultivar. The water and salt stresses as well as the disintegration of leaf tissue on the contrary altered cytochrome oxidase from the oxidized to its reduced state in CO-insensitive cultivars; probably by changing the cellular integrity, turgidity and structure of mitochondrial membrane, and also due to decreased mitochondrial energization.

  3. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  4. Cloning and sequence of the human adrenodoxin reductase gene.

    PubMed Central

    Lin, D; Shi, Y F; Miller, W L

    1990-01-01

    Adrenodoxin reductase (ferrodoxin:NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. We cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G + C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of "housekeeping" genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon. Images PMID:2236061

  5. A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae.

    PubMed

    Hopper, Amanda C; Li, Ying; Cole, Jeffrey A

    2013-06-01

    Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae. PMID:23543713

  6. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  7. Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.

    PubMed

    de Boer, A P; van der Oost, J; Reijnders, W N; Westerhoff, H V; Stouthamer, A H; van Spanning, R J

    1996-12-15

    The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower

  8. Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

    PubMed

    Zhang, W; Parentau, H; Greenly, R L; Metz, C A; Aggarwal, S; Wainer, I W; Tracy, T S

    1999-01-01

    Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.

  9. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

  10. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  11. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  12. Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

    PubMed

    Ewen, Kerstin Maria; Hannemann, Frank; Khatri, Yogan; Perlova, Olena; Kappl, Reinhard; Krug, Daniel; Hüttermann, Jürgen; Müller, Rolf; Bernhardt, Rita

    2009-10-16

    Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity. PMID:19696019

  13. Diversification of catalytic function in a synthetic family of chimeric cytochrome p450s.

    PubMed

    Landwehr, Marco; Carbone, Martina; Otey, Christopher R; Li, Yougen; Arnold, Frances H

    2007-03-01

    We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping. PMID:17379142

  14. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  15. Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus.

    PubMed Central

    Burke, K A; Lascelles, J

    1976-01-01

    Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase. PMID:1262303

  16. Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria

    SciTech Connect

    Oftebro, H.; Saarem, K.; Bjoerkhem, I.; Pedersen, J.I.

    1981-11-01

    The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation on bile acid intermediates and in the 25-hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mitochondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of C27-steroids and 25-hydroxylation of vitamin D3 when reconstructed with NADPH, the ferredoxin and the ferredoxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450(-1) X min-1) were found: cholesterol, 8; 5-cholestene-3 beta, 7 alpha-diol, 10; 7 alpha-hydroxy-4-cholesten-3-one, 23; 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one, 27; 5 beta-cholestane-3 alpha, 7 alpha-diol, 28; 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyrapone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferredoxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase. It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of C27-steroids and vitamin D3.

  17. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

  18. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  19. N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation.

    PubMed Central

    Moore, G R; Cox, M C; Crowe, D; Osborne, M J; Rosell, F I; Bujons, J; Barker, P D; Mauk, M R; Mauk, A G

    1998-01-01

    The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes. PMID:9601073

  20. Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

    SciTech Connect

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio . E-mail: n-shimamoto@kph.bunri-u.ac.jp

    2006-07-15

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as {alpha}-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

  1. Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whale (Delphinapterus leucas).

    PubMed

    White, R D; Hahn, M E; Lockhart, W L; Stegeman, J J

    1994-05-01

    Understanding the effects of environmental contaminants on cetaceans and other marine mammals will require information on the biochemistry of xenobiotic metabolism in these species. We characterized the hepatic microsomal cytochrome P450 system in beluga whales (Delphinapterus leucas) from the Canadian Arctic. The content of native P450 averaged 0.203 and 0.319 nmol/mg microsomal protein, cytochrome b5 content averaged 0.199 and 0.236 nmol/mg, and rates of NADPH-cytochrome c reductase were 79 and 76 nmol/min/mg, for females and males respectively. Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), and benzo[a]pyrene (BP) hydroxylase (AHH) activities were significantly greater in males than in females, and were highly correlated with one another (r2 between 0.853 and 0.912). HPLC analysis of in vitro BP metabolites revealed benzo-ring (7,8- and 9,10-) dihydrodiols, consistent with activation of this compound, as well as 4,5-dihydrodiol,3-OH-, 7-OH-, and 9-OH-BP and 1,6- and 3,6-quinones. Estradiol 2-hydroxylase activity did not differ between sexes, and rates did not correlate with those of the other activities. Antibodies against scup P450B (an apparent teleost CYP2B) and rat CYP2B1 did not recognize proteins in beluga liver microsomes, but there was a protein detected by antibodies to PB-inducible rabbit CYP2B4. Antibodies to ethanol and ketone-inducible rat CYP2E1 reacted with two proteins in beluga liver microsomes. Antibodies specific to hydrocarbon-inducible CYP1A1 and/or CYP1A2 forms showed a single protein band, apparently more closely related to CYP1A1. The content of CYP1A was fivefold greater in male than in female beluga. CYP1A content was highly correlated with EROD, PROD, and AHH activities, suggesting that this P450 form is a primary catalyst for these reactions in beluga. CYP1A content and activity were highly correlated with the concentrations in blubber of non-ortho and mono-ortho PCB congeners, compounds that induce CYP1A

  2. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Temporary Department of State press building passes. 9b.5 Section 9b.5 Foreign Relations DEPARTMENT OF STATE GENERAL REGULATIONS GOVERNING DEPARTMENT OF STATE PRESS BUILDING PASSES § 9b.5 Temporary Department of State press building passes. A...

  3. 17 CFR 240.12b-5 - Determination of affiliates of banks.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... banks. 240.12b-5 Section 240.12b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Securities Exchange Act of 1934 General § 240.12b-5 Determination of affiliates of banks. In determining whether a person is an “affiliate” or “parent” of a bank or whether a bank is a “subsidiary” or...

  4. 26 CFR 301.7701(b)-5 - Coordination with section 877.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Coordination with section 877. 301.7701(b)-5 Section 301.7701(b)-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) PROCEDURE AND ADMINISTRATION PROCEDURE AND ADMINISTRATION Definitions § 301.7701(b)-5 Coordination...

  5. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers

    PubMed Central

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H.; Frei, Eva; Arlt, Volker M.; White, P.

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5, to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP‐7,8‐dihydrodiol and BaP‐9‐ol, which are intermediates in BaP‐derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP‐3‐ol, a metabolite that is a ‘detoxified’ product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP‐7,8‐dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP‐9‐ol. BaP‐3‐ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229–235, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26919089

  6. Catalytic and Immunochemical Detection of Hepatic and Extrahepatic Microsomal Cytochrome P450 1A1 (CYP1A1) in White-sided Dolphin (Lagenorhynchus acutus)

    PubMed Central

    Wilson, Joanna Y.; Moore, Michael J.; Stegeman, John J.

    2009-01-01

    We have characterized microsomal systems and measured the levels of microsomal cytochrome P450 1A1 (CYP1A1) and ethoxyresorufin-O-deethylase activity in multiple internal organs of male and female white-sided dolphin (Lagenorhynchus acutus) from the northwest Atlantic Ocean. Internal organs were sampled within 24 hours of death, sometimes in a period of hours, collection times which are significantly less than usually seen for marine mammals. Tissue autolysis, as assessed by histological analysis of liver, was minimal to none in all individuals. Total P420 did not correlate with time from death to sampling, suggesting that it is a poor indicator of P450 degradation in cetacean tissues where perfusion isn’t practical. The total hepatic microsomal P450 content, cytochrome b5 content, and NADPH-cytochrome c (P450) reductase (CPR) activity averaged 0.29 nmol mg−1, 0.12 nmol mg−1, and 238 nmol mg−1 min−1, respectively. Microsomal CPR activity in liver was higher than that in lung and kidney, and was higher than that reported in liver of most other cetacean species. Immunodetected CYP1A1 content was low in all organs, less than 3 pmoles CYP1A equivalents mg−1. EROD activity ranged from 9 – 376 pmoles mg−1 min−1 and was greater in liver than in other tissues. Hepatic microsomal EROD activity and CYP1A1 content did not correlate. However, hepatic EROD activity, but not CYP1A1 protein content, was well correlated with both total PCB and Σmono-ortho PCB concentrations in blubber. Length, as a proxy for age, did not correlate with hepatic EROD activity or CYP1A1 protein levels, and sex did not influence the relationship between EROD and contaminant concentrations. We cannot easily control for the extent of tissue degradation in cetacean studies nor do we have a complete history of these animals. Therefore, other factors such as degradation or hormonal state may have a role in the observed relationships. Yet, as in other mammals, hepatic tissues appear to be

  7. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  8. Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

    PubMed

    Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

    2012-01-01

    Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa. PMID:22297231

  9. Ribonucleotide Reductase-- a Radical Enzyme

    NASA Astrophysics Data System (ADS)

    Reichard, Peter; Ehrenberg, Anders

    1983-08-01

    Ribonucleotide reductases catalyze the enzymatic formation of deoxyribonucleotides, an obligatory step in DNA synthesis. The native form of the enzyme from Escherichia coli or from mammalian sources contains as part of its polypeptide structure a free tyrosyl radical, stabilized by an iron center. The radical participates in all probability in the catalytic process during the substitution of the hydroxyl group at C-2 of ribose by a hydrogen atom. A second, inactive form of the E. coli reductase lacks the tyrosyl radical. Extracts from E. coli contain activities that interconvert the two forms. The tyrosyl radical is introduced in the presence of oxygen, while anaerobiosis favors its removal, suggesting a regulatory role in DNA synthesis for oxygen.

  10. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  11. The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification.

    PubMed

    Carr, G J; Page, M D; Ferguson, S J

    1989-02-15

    1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an

  12. Flower colour and cytochromes P450†

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  13. Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

    PubMed Central

    Shalloe, F; Elliott, G; Ennis, O; Mantle, T J

    1996-01-01

    A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose. PMID:8687377

  14. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  15. Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis.

    PubMed

    Takubo, Keiko; Morikawa, Tomomi; Nonaka, Yasuki; Mizutani, Masaharu; Takenaka, Shigeo; Takabe, Keiji; Takahashi, Masa-aki; Ohta, Daisaku

    2003-07-01

    We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis. Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin. First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively. The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c in vitro. Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR. RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower. Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.

  16. Design and improvement of artificial redox modules by molecular fusion of flavodoxin and flavodoxin reductase from Escherichia coli

    PubMed Central

    Bakkes, Patrick J.; Biemann, Stefan; Bokel, Ansgar; Eickholt, Marc; Girhard, Marco; Urlacher, Vlada B.

    2015-01-01

    A variety of fusion proteins between the versatile redox partners flavodoxin (FldA) and flavodoxin reductase (Fpr) from Escherichia coli was constructed with the aim to improve the electron transfer properties. The order in which FldA and Fpr were fused and the linker region between them was varied in a systematic manner. A simple molecular tool, designated “DuaLinX”, was developed that facilitated the parallel introduction of flexible glycine-rich and rigid proline-rich linkers between the fusion partners in a single cloning event. The fusion constructs were tested for their ability to transfer electrons to cytochrome c and cytochrome P450 109B1 from Bacillus subtilis. With CYP109B1, the performance of the constructs showed, independent of the domain order, a strong dependency on linker length, whereas with cytochrome c this phenomenon was less pronounced. Constructs carrying linkers of ≥15 residues effectively supported the CYP109B1-catalysed hydroxylation of myristic acid. Constructs carrying proline-rich linkers generally outperformed their glycine-rich counterparts. The best construct, FldA-Fpr carrying linker ([E/L]PPPP)4, supported CYP109B1 activity equally well as equivalent amounts of the non-fused redox partners, while cytochrome c reductase activity was ~2.7-fold improved. Thus, to functionally connect redox partners, rigid proline-rich linkers may be attractive alternatives to the commonly used flexible glycine-rich linkers. PMID:26177696

  17. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    PubMed Central

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

    2013-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

  18. Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole. Isolation of an NN-bridged benzyne-protoporphyrin IX adduct.

    PubMed

    Ortiz de Montellano, P R; Mathews, J M

    1981-06-01

    Destruction of hepatic cytochrome P-450 during catalytic processing of 1-amino-benzotriazole is accompanied by an equal loss of microsomal haem but not by loss of cytochrome b5, or stimulation of lipid peroxidation. An abnormal porphyrin, tentatively identified as an NN-bridged benzyne-protoporphyrin IX adduct, appears to be formed by the addition of catalytically generated benzyne to prosthetic haem.

  19. Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

    This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

  20. 296-B-5 Stack monitoring and sampling system annual system assessment report

    SciTech Connect

    Ridge, T.M.

    1995-02-01

    The B Plant Administration Manual requires an annual system assessment to evaluate and report the present condition of the sampling and monitoring system associated with Stack 296-B-5 at B Plant. The sampling and monitoring system associated with stack 296-B-5 is functional and performing satisfactorily. This document is an annual assessment report of the systems associated with the 296-B-5 stack.

  1. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  2. The Cox3p assembly module of yeast cytochrome oxidase

    PubMed Central

    Su, Chen-Hsien; McStay, Gavin P.; Tzagoloff, Alexander

    2014-01-01

    Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome. PMID:24478450

  3. Biological diversity of cytochrome P450 redox partner systems.

    PubMed

    McLean, Kirsty J; Luciakova, Dominika; Belcher, James; Tee, Kang Lan; Munro, Andrew W

    2015-01-01

    Cytochrome P450 enzymes (P450s or CYPs) catalyze an enormous variety of oxidative reactions in organisms from all major domains of life. Their monooxygenase activity relies on the reductive scission of molecular oxygen (O2) bound to P450 heme iron, and thus on the delivery of two electrons to the heme iron at discrete points in the catalytic cycle. Early studies suggested that P450 redox partner machinery fell into only two major classes: either the eukaryotic diflavin enzyme NADPH-cytochrome P450 oxidoreductase, or bacterial/mitochondrial NAD(P)H-ferredoxin reductase and ferredoxin partners. However, more recent studies, aided by genome sequence data, reveal a much more complex scenario. Several new types of P450 redox partner systems have now been characterized, including P450s naturally linked to their redox partners, or to a component protein of their P450 electron delivery system. Other P450s have evolved to bypass requirements for redox partners, and instead react directly with hydrogen peroxide or NAD(P)H to facilitate oxidative or reductive catalysis. Further P450s are fused to non-redox partner enzymes and can catalyse consecutive reactions in a common pathway. This chapter describes the biochemistry and the enormous natural diversity of P450 redox systems, including descriptions of novel P450s fused to non-redox partner proteins.

  4. Kinetic and spectroscopic studies of cytochrome b-563 in isolated cytochrome b/f complex and in thylakoid membranes

    SciTech Connect

    Hind, G.; Clark, R.D.; Houchins, J.P.

    1983-01-01

    Extensive studies, performed principally by Hauska, Hurt and collaborators, have shown that a cytochrome (cyt) b/f complex isolated from photosynthetic membranes of spinach or Anabaena catalyzes electron transport from plastoquinol (PQH/sub 2/) to plastocyanin or algal cyt c-552. The complex from spinach thylakoids generated a membrane potential when reconstituted into liposomes, and although the electrogenic mechanism remains unknown, a key role for cyt b-563 is widely accepted. Electrogenesis by a Q-cycle mechanism requires a plastoquinone (PQ) reductase to be associated with the stromal side of the thylakoid b/f complex though this activity has yet to be demonstrated. It seemed possible that more gentle isolation of the complex might yield a form containing additional polypeptides, perhaps including a PQ reductase or a component involved in returning electrons from reduced ferredoxin to the complex in cyclic electron flow. Optimization of the isolation of cyt b/f complex for Hybrid 424 spinach from a growth room was also required. The procedure we devised is compared to the protocol of Hurt and Hauska (1982). 13 references.

  5. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  6. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  7. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

    PubMed

    Tamazato Longhi, Mariana; Magalhães, Magna; Reina, Jeffrey; Morais Freitas, Vanessa; Cella, Nathalie

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  8. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell. PMID:22028781

  9. Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana1

    PubMed Central

    Amy, Nancy K.; Garrett, Reginald H.

    1974-01-01

    The assimilatory nitrate reductase (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated. Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity. NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction. Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme. The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal

  10. External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

    PubMed

    Nikiforova, Anna B; Saris, Nils-Erik L; Kruglov, Alexey G

    2014-09-01

    It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane. PMID:24945955

  11. Testosterone induction of microsomal acyl-CoA reductase and a cytosolic regulatory protein in mouse preputial glands.

    PubMed

    Lee, T C; Kirk, P; Snyder, F

    1986-01-01

    Alkyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids are prominent components of many mammalian cells; moreover, an acetylated form of an alkyl phospholipid was recently found to possess potent hypotensive, inflammatory and allergic properties. In our studies, preputial glands of mice were selected as a model to investigate the regulation of factors involved in the biosynthesis of ether-linked lipids, since these glands contain high concentrations of ether-linked neutral lipids that are under the influence of hormonal control. We found that a key enzyme in the ether-lipid metabolic pathway, microsomal acyl-CoA reductase that catalyzes the formation of long-chain fatty alcohols (precursor of the O-alkyl chain), was increased 16-fold after injecting testosterone into male, castrated mice. This induction was highly specific, since testosterone did not affect another microsomal enzyme, NADPH-cytochrome c reductase. Based on kinetics of enzyme activity changes, the half-life of acyl-CoA reductase was calculated to be 61-70 h. In addition, the activity of a cytosolic stimulatory protein for the acyl-CoA reductase (but not for a different cytosolic protein, lactate dehydrogenase) was also enhanced in the testosterone-treated, male, castrated mice. These findings indicate that acyl-CoA reductase is an important regulatory enzyme in the reactions that lead to the formation of the ether bond in glycerolipids and that it is modulated through hormonal control. PMID:3940533

  12. TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.

    PubMed

    Han, Dohyun; Kim, Kyunggon; Oh, Jongkil; Park, Jungeun; Kim, Youngsoo

    2008-02-15

    Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

  13. New functional sulfide oxidase-oxygen reductase supercomplex in the membrane of the hyperthermophilic bacterium Aquifex aeolicus.

    PubMed

    Prunetti, Laurence; Infossi, Pascale; Brugna, Myriam; Ebel, Christine; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2010-12-31

    Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H(2)S) to molecular oxygen. H(2)S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc(1) complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c(555) and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba(3)-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O(2) in the presence of the electron donor, H(2)S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.

  14. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus LBB.B5

    PubMed Central

    Hajo, Karima; Lenoci, Leonardo; Bron, Peter A.; Dijkstra, Annereinou; Alkema, Wynand; Wels, Michiel; Siezen, Roland J.; Minkova, Svetlana; van Hijum, Sacha A. F. T.

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus. The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus. PMID:27795256

  15. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  16. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  17. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  18. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  19. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  20. 26 CFR 1.410(b)-5 - Average benefit percentage test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Average benefit percentage test. 1.410(b)-5...) INCOME TAX (CONTINUED) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.410(b)-5 Average benefit percentage test. (a) General rule. A plan satisfies the average benefit percentage test of...

  1. 40 CFR Table B-5 to Subpart B of... - Symbols and Abbreviations

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 6 2013-07-01 2013-07-01 false Symbols and Abbreviations B Table B-5 to Subpart B of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Performance Characteristics of Automated Methods for SO2, CO, O3, and NO2 Pt. 53, Subpt. B, Table B-5 Table...

  2. Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450.

    PubMed

    Stevenson, P M; Ruettinger, R T; Fulco, A J

    1983-05-16

    Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450. PMID:6405752

  3. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    SciTech Connect

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

  4. Photo-initiated crosslinking extends mapping of the protein-protein interface to membrane-embedded portions of cytochromes P450 2B4 and b₅.

    PubMed

    Ječmen, Tomáš; Ptáčková, Renata; Černá, Věra; Dračínská, Helena; Hodek, Petr; Stiborová, Marie; Hudeček, Jiří; Šulc, Miroslav

    2015-11-01

    Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We

  5. Separation, purification, and properties of cytochrome P-450 from uninduced rat liver microsomes for the studies of metabolism of environmental chemicals

    SciTech Connect

    Dialameh, G.H. )

    1988-09-01

    This study reports the authors present results on the development of a procedure for purification of multiple forms of cytochrome P-450 from un-induced rat liver microsomes. These cytochromes are catalytically active when reconstituted with NADPH-cytochrome c reductase and lipid and exhibit substrate specificities. The presence of four distinct forms of cytochrome P-450 in uninduced rat liver microsomes which is the result of this research report, compared with the presence of six forms in induced animals represent the importance of genetic control of these enzymes for the metabolism and detoxification of environmental chemicals. These metabolite patterns are not only different for the various species, but also among different individuals. The molecular basis for this are genetic and environmental factors, which exhibit interesting evolutionary aspects.

  6. Stability, redox parameters and electrocatalytic activity of a cytochrome domain from a new subfamily.

    PubMed

    Molinas, María F; Benavides, Leandro; Castro, María A; Murgida, Daniel H

    2015-10-01

    We report a spectroscopic, electrochemical and spectroelectrochemical characterization of the soluble cytochrome c domain (Cyt-D) from the Rhodothermus marinus caa3 terminal oxygen reductase and its putative electron donor, a high potential [4Fe-4S] protein (HiPIP). Cyt-D exhibits superior stability, particularly at the level of the heme pocket, compared to archetypical cytochromes in terms of thermal and chemical denaturation, alkaline transition and oxidative bleaching of the heme, which is further increased upon adsorption on biomimetic electrodes. Therefore, this protein is proposed as a suitable building block for electrochemical biosensing. As a proof of concept, we show that the immobilized Cyt-D exhibits good electrocatalytic activity towards H2O2 reduction. Relevant thermodynamic and kinetic electron transfer parameters for Cyt-D and HiPIP are also reported, including reorganization energies of 0.33 eV and 0.42 eV, respectively.

  7. Cytochromes and iron sulfur proteins in sulfur metabolism of phototrophic bacteria

    NASA Technical Reports Server (NTRS)

    Fischer, U.

    1985-01-01

    Dissimilatory sulfur metabolism in phototrophic sulfur bacteria provides the bacteria with electrons for photosynthetic electron transport chain and, with energy. Assimilatory sulfate reduction is necessary for the biosynthesis of sulfur-containing cell components. Sulfide, thiosulfate, and elemental sulfur are the sulfur compounds most commonly used by phototrophic bacteria as electron donors for anoxygenic photosynthesis. Cytochromes or other electron transfer proteins, like high-potential-iron-sulfur protein (HIPIP) function as electron acceptors or donors for most enzymatic steps during the oxidation pathways of sulfide or thiosulfate. Yet, heme- or siroheme-containing proteins themselves undergo enzymatic activities in sulfur metabolism. Sirohemes comprise a porphyrin-like prosthetic group of sulfate reductase. eenzymatic reactions involve electron transfer. Electron donors or acceptors are necessary for each reaction. Cytochromes and iron sulfur problems, are able to transfer electrons.

  8. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

    Pechurskaya, Tatiana A. . E-mail: usanov@iboch.bas-net.by

    2007-02-16

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.

  9. Biliverdin reductase isozymes in metabolism.

    PubMed

    O'Brien, Luke; Hosick, Peter A; John, Kezia; Stec, David E; Hinds, Terry D

    2015-04-01

    The biliverdin reductase (BVR) isozymes BVRA and BVRB are cell surface membrane receptors with pleiotropic functions. This review compares, for the first time, the structural and functional differences between the isozymes. They reduce biliverdin, a byproduct of heme catabolism, to bilirubin, display kinase activity, and BVRA, but not BVRB, can act as a transcription factor. The binding motifs present in the BVR isozymes allow a wide range of interactions with components of metabolically important signaling pathways such as the insulin receptor kinase cascades, protein kinases (PKs), and inflammatory mediators. In addition, serum bilirubin levels have been negatively associated with abdominal obesity and hypertriglyceridemia. We discuss the roles of the BVR isozymes in metabolism and their potential as therapeutic targets. PMID:25726384

  10. Regulation of human adipogenesis by miR125b-5p.

    PubMed

    Rockstroh, Denise; Löffler, Dennis; Kiess, Wieland; Landgraf, Kathrin; Körner, Antje

    2016-01-01

    MicroRNAs (miRNAs) are non-coding RNAs that regulate target gene expression at the post-transcriptional level and are supposed to be implicated in the control of adipogenesis. We aimed to identify miRNAs which are involved in the regulation of human adipogenesis and searched for their molecular targets. Applying microarray-analysis we identified miR125b-5p as upregulated during human adipocyte differentiation, although its role during adipogenesis is unknown. We identified and characterized the matrix metalloproteinase 11 (MMP11) as a direct target of miR125b-5p by showing that miR125b-5p overexpression significantly reduces MMP11 luciferase activity and mutation of any single binding site was sufficient to abolish the miR125b-5p mediated inhibition of luciferase activity. MMP11 overexpression decreased fat accumulation, indicating that MMP11 acts as an anti-adipogenic regulator. In contrast, overexpression of miR125b-5p itself reduced adipogenesis. In summary, we identified miR125b-5p as upregulated during human adipogenesis indicating that miR125b-5p may serve as a regulator of human adipocyte differentiation. We further show that miR125b-5p downregulates the anti-adipogenic MMP11, but directly inhibits adipogenesis itself. Taken together, these data implicate that miR125b-5p can affect human adipogenesis via MMP11 and probably additional targets. PMID:27617174

  11. Regulation of human adipogenesis by miR125b-5p

    PubMed Central

    Rockstroh, Denise; Löffler, Dennis; Kiess, Wieland; Landgraf, Kathrin; Körner, Antje

    2016-01-01

    ABSTRACT MicroRNAs (miRNAs) are non-coding RNAs that regulate target gene expression at the post-transcriptional level and are supposed to be implicated in the control of adipogenesis. We aimed to identify miRNAs which are involved in the regulation of human adipogenesis and searched for their molecular targets. Applying microarray-analysis we identified miR125b-5p as upregulated during human adipocyte differentiation, although its role during adipogenesis is unknown. We identified and characterized the matrix metalloproteinase 11 (MMP11) as a direct target of miR125b-5p by showing that miR125b-5p overexpression significantly reduces MMP11 luciferase activity and mutation of any single binding site was sufficient to abolish the miR125b-5p mediated inhibition of luciferase activity. MMP11 overexpression decreased fat accumulation, indicating that MMP11 acts as an anti-adipogenic regulator. In contrast, overexpression of miR125b-5p itself reduced adipogenesis. In summary, we identified miR125b-5p as upregulated during human adipogenesis indicating that miR125b-5p may serve as a regulator of human adipocyte differentiation. We further show that miR125b-5p downregulates the anti-adipogenic MMP11, but directly inhibits adipogenesis itself. Taken together, these data implicate that miR125b-5p can affect human adipogenesis via MMP11 and probably additional targets. PMID:27617174

  12. 5 alpha-reductase deficiency without hypospadias.

    PubMed Central

    Ng, W K; Taylor, N F; Hughes, I A; Taylor, J; Ransley, P G; Grant, D B

    1990-01-01

    A boy aged 4 with penoscrotal hypospadias and his brother aged 12 with micropenis had typical changes of homozygous 5 alpha-reductase deficiency. After three injections of chorionic gonadotrophin there was a trivial rise in plasma dihydrotestosterone with a normal increase in plasma testosterone. Urine steroid chromatography showed abnormally high 5 beta: 5 alpha ratios and 5 alpha-reductase activity was appreciably reduced in genital skin fibroblasts. The results indicate that 5 alpha-reductase deficiency is not invariably associated with genital ambiguity. PMID:2248513

  13. A Common Variant Of Ubiquinol-Cytochrome c Reductase Complex Is Associated with DDH

    PubMed Central

    Dai, Jin; Chen, Dongyang; Xu, Zhihong; Shi, Dongquan; Mao, Ping; Teng, Huajian; Gao, Xiang; Hu, Zhibin; Shen, Hongbing; Jiang, Qing

    2015-01-01

    Purpose Genetic basis of Developmental dysplasia of the hip (DDH) remains largely unknown. To find new susceptibility genes for DDH, we carried out a genome-wide association study (GWAS) for DDH. Methods We enrolled 386 radiology confirmed DDH patients and 558 healthy controls (Set A) to conduct a genome-wide association study (GWAS). Quality-control was conducted at both the sample and single nucleotide polymorphism (SNP) levels. We then conducted a subsequent case-control study to replicate the association between a promising loci, rs6060373 in UQCC gene and DDH in an independent set of 755 cases and 944 controls (set B). Results In the DDH GWAS discovering stage, 51 SNPs showed significance of less than 10-4, and another 577 SNPs showed significance of less than 10-3. In UQCC, all the 12 genotyped SNPs showed as promising risk loci. Genotyping of rs6060373 in set A showed the minor allele A as a promising risk allele (p = 4.82*10-7). In set A, the odds ratio of allele A was 1.77. Genotyping of rs6060373 in Set B produced another significant result (p = 0.0338) with an odds ratio of 1.18 for risk allele A. Combining set A and set B, we identified a total p value of 3.63*10-6 with the odds ratio of 1.35 (1.19–1.53) for allele A. Conclusion Our study demonstrates common variants of UQCC, specifically rs6060373, are associated with DDH in Han Chinese population. PMID:25848760

  14. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains

    SciTech Connect

    Crawford, N.M.; Smith, M.; Bellissimo, D.; Davis, R.W. )

    1988-07-01

    The sequence of nitrate reductase mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a {lambda}gt10 cDNA library of Arabidopsis leaf poly(A){sup +} RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being >80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b{sub 5} superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b{sub 5} reductase.

  15. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  16. Genetics Home Reference: sepiapterin reductase deficiency

    MedlinePlus

    ... reductase enzyme. This enzyme is involved in the production of a molecule called tetrahydrobiopterin (also known as ... is responsible for the last step in the production of tetrahydrobiopterin. Tetrahydrobiopterin helps process several building blocks ...

  17. Synthesis of NaB5C bulk ceramics by reaction sintering

    NASA Astrophysics Data System (ADS)

    Morito, Haruhiko; Anzai, Jun; Kimura, Takuma; Yamane, Hisanori

    2015-09-01

    Bulk ceramics of NaB5C were prepared by heating compact bodies of amorphous boron (B) and carbon black (C) powders with Na at 1073 K. The obtained bulk ceramics retained the rectangular shape of their original compacts. The obtained samples had a density of 80.1 ± 0.6% of the theoretical density of NaB5C. NaB5C bulk ceramics were also prepared by heating compacts comprised of B and C powders and Na. The addition of Na to the starting compact bodies increased the relative bulk density to 83.5 ± 0.4%. A fracture bending strength of 195 MPa was measured for the NaB5C bulk sample prepared from the compact of Na, B, and C.

  18. 3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp.

    PubMed Central

    Sheldon, P S; Kekwick, R G; Sidebottom, C; Smith, C G; Slabas, A R

    1990-01-01

    The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp. Images Fig. 2. Fig. 5. Fig. 6. PMID:2244875

  19. Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.

    PubMed Central

    Edwards, A M; Blasie, J K; Bean, J C

    1998-01-01

    Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase. PMID:9512031

  20. Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

    PubMed

    Lorenzo, María M; Sánchez-Puig, Juana M; Blasco, Rafael

    2012-04-01

    The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

  1. A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

    PubMed

    Winter, J; Eckerskorn, C; Waditschatka, R; Kayser, H

    2001-11-01

    A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450.

  2. A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

    PubMed

    Winter, J; Eckerskorn, C; Waditschatka, R; Kayser, H

    2001-11-01

    A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450. PMID:11767943

  3. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  4. A possible role of Aspergillus niger mitochondrial cytochrome c in malachite green reduction under calcium chloride stress.

    PubMed

    Gomaa, Ola M; Selim, Nabila S; Linz, John E

    2013-01-01

    In previous work, decolorization of malachite green (MG) was studied in Aspergillus niger in the presence and absence of calcium chloride stress. Decolorization took place within 24 h, and a signal transduction process that initiated MG decolorization was suggested to be involved. In the present study, further investigation of the relationship between calcium chloride stress and enhanced MG biodegradation was conducted at the sub-cellular level. MG-NADH reductase activity, a key enzyme in MG decolorization, was produced as decolorization commenced, and enzyme activity increased threefold upon exposure to calcium chloride. Inhibitors of cytochrome p450, Ca(2+) channel activity as well as activity of the signaling protein phosphoinositide 3-kinase were tested. All three activities were inhibited to different extents resulting in reduced MG decolorization. Spectral analysis of the mitochondrial fraction showed a heme signal at 405 nm and A405/A280 ratio that is characteristic of the porphoryin ring of cytochromes. There were no peaks detected for cytochromes a or b, but a shoulder appearing at 550 nm was observed, which suggested that cytochrome c is involved; the absorbance for cytochrome c doubled after calcium chloride stress supporting this idea. MG decolorization took place via a series of demethylation steps, and cytotoxicity analysis revealed a decrease in the toxicity associated with generation of leucomalachite green.

  5. Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice.

    PubMed

    Kunz, W S; Kuznetsov, A V; Clark, J F; Tracey, I; Elger, C E

    1999-04-01

    Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.

  6. Canine cytochrome P-450 pharmacogenetics.

    PubMed

    Court, Michael H

    2013-09-01

    The cytochrome P-450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences.

  7. Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

    SciTech Connect

    Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

    1984-10-01

    The hepatic cytochrome P-450-mediated metabolism and metabolic activation of (chloroethyl-3H)cyclophosphamide (( chloroethyl-3H)CP) and (4-14C)cyclophosphamide (( 4-14C)CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of (14C)acrolein, a metabolite of (4-14C)CP, were also investigated. The metabolism of (chloroethyl-3H)CP and (4-14C)CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between (4-14C)CP and (chloroethyl-3H)CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of (chloroethyl-3H)CP to nucleic acids and almost exclusive binding of (4-14C)CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with (4-14C)CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with (14C)acrolein in the presence and the absence of NADPH. The results confirmed covalent association between (14C)acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of (14C)acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations.

  8. The roles of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f.

    PubMed

    Wood, P M

    1977-02-01

    A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.

  9. The aldo-keto reductase superfamily homepage.

    PubMed

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  10. Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens.

    PubMed Central

    Morris, C J; Black, A C; Pealing, S L; Manson, F D; Chapman, S K; Reid, G A; Gibson, D M; Ward, F B

    1994-01-01

    The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low

  11. Modeling and signal analysis of semiconducting B(5)C neutron detectors

    NASA Astrophysics Data System (ADS)

    Harken, Andrew D.

    Neutron detectors are needed for a myriad of applications ranging from military uses to power generation monitors to medical radiation therapy. Recently, a class of semiconducting boron carbide (B5C)/silicon heterojunction diodes were demonstrated to detect thermal neutrons.[1] The B5C-based devices have advantageous features of requiring low operating voltage, low power, are robust and extremely thin while maintaining detection efficiency. A simple model was developed for the analysis of the neutron capture output spectrum from the detectors, which allowed the comparison of several differing styles of planar geometry detectors. The model was also utilized to obtain the functional dependence of the device efficiencies, capture product spectral features, and the capture product energy deposition on capture layer thickness. An all-B5C device construction was determined by the model to be the most efficient form of a B5C-based detector, which reaches nearly 100% detection efficiency with a low probability of false positives. This model showed agreement with output from a full-physics simulation package, GEANT4, and experimental neutron detection spectra from a B5C/Si device. The signals generated in a B5C/Si heterojunction diode during neutron and alpha particle detection experiments were analyzed through fitting of the output current pulses and through capture output spectra. The output current pulse analysis confirmed charge generation and collection from both materials in the diode and demonstrated the suitability of the B5C material for use in an all-semiconducting B5C neutron detector. The experimental output spectra were analyzed and determined to be lower in detected capture product energy than expected, but retained the spectral features that allowed analysis of the detection results. The development of the model and the results from the particle detection experiments show great promise for the future development of B5C neutron detectors. [1]B. W. Robertson, S

  12. MicroRNA-125b-5p mimic inhibits acute liver failure.

    PubMed

    Yang, Dakai; Yuan, Qinggong; Balakrishnan, Asha; Bantel, Heike; Klusmann, Jan-Henning; Manns, Michael P; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep

    2016-01-01

    The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF. PMID:27336362

  13. MicroRNA-125b-5p mimic inhibits acute liver failure

    PubMed Central

    Yang, Dakai; Yuan, Qinggong; Balakrishnan, Asha; Bantel, Heike; Klusmann, Jan-Henning; Manns, Michael P.; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep

    2016-01-01

    The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF. PMID:27336362

  14. Crystallization of Mitochondrial Cytochrome Oxidase

    NASA Astrophysics Data System (ADS)

    Ozawa, Takayuki; Tanaka, Masashi; Wakabayashi, Takashi

    1982-12-01

    Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 μ m in average length and 5 μ m in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 angstrom, b = 84 angstrom, and c = 103 angstrom, and α =β 90 degrees, γ = 126 degrees.

  15. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  16. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  17. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6.

    PubMed

    Sridar, Chitra; Kent, Ute M; Notley, Lisa M; Gillam, Elizabeth M J; Hollenberg, Paul F

    2002-06-01

    Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity. PMID:12023523

  18. Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

    PubMed

    Schroedl, N A; Hartzell, C R

    1977-11-15

    Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.

  19. Ajoene is an inhibitor and subversive substrate of human glutathione reductase and Trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies.

    PubMed

    Gallwitz, H; Bonse, S; Martinez-Cruz, A; Schlichting, I; Schumacher, K; Krauth-Siegel, R L

    1999-02-11

    Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.

  20. Cytochrome a1 of acetobacter aceti is a cytochrome ba functioning as ubiquinol oxidase.

    PubMed

    Matsushita, K; Shinagawa, E; Adachi, O; Ameyama, M

    1990-12-01

    Cytochrome a1 is a classic cytochrome that in the 1930s had already been detected in Acetobacter strains and in the 1950s was identified as a terminal oxidase. However, recent studies did not substantiate the previous observations. We have detected a cytochrome a1-like chromophore in Acetobacter aceti, which was purified and characterized in this study. The cytochrome was solubilized from membranes of the strain with octyl beta-D-glucopyranoside and was purified by single column chromatography. The purified cytochrome exhibited a broad alpha peak around 600-610 nm, which turned to a sharp peak at 589 nm in the presence of cyanide. Carbon monoxide difference spectra of the cytochrome indicated the presence of an alpha-type cytochrome. The cytochrome contained 1 mol each of hemes b and a and probably one copper ion. These results suggest that the cytochrome purified from A. aceti is the so-called cytochrome a1, and thus the existence of the classic cytochrome has been reconfirmed. The purified enzyme consisted of four polypeptides of 55, 35, 22, and 18 kDa, and it showed a sedimentation coefficient of 6.3 S in the native form. The enzyme had a high ubiquinol oxidase activity (140-160 mumol of ubiquinol-2 oxidized per min per mg of protein). When reconstituted into proteoliposomes, the cytochrome could generate an electrochemical proton gradient during oxidation of ubiquinol. Thus, cytochrome a1 of A. aceti has been shown to be a cytochrome ba terminal oxidase capable of generating an electrochemical proton gradient concomitant with ubiquinol oxidation.

  1. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  2. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; Hoeft, Shelley E.; Oremland, Ronald S.; Basu, Partha; Stolz, John F.

    2009-05-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  3. 26 CFR 20.2056(b)-5 - Marital deduction; life estate with power of appointment in surviving spouse.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... appointment in surviving spouse. 20.2056(b)-5 Section 20.2056(b)-5 Internal Revenue INTERNAL REVENUE SERVICE... surviving spouse. (a) In general. Section 2056(b)(5) provides that if an interest in property passes from the decedent to his surviving spouse (whether or not in trust) and the spouse is entitled for life...

  4. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    PubMed

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  5. Construction and engineering of a thermostable self-sufficient cytochrome P450

    SciTech Connect

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  6. Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

    PubMed Central

    Cramm, R; Siddiqui, R A; Friedrich, B

    1997-01-01

    Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification. PMID:9352929

  7. 26 CFR 1.50B-5 - Limitations with respect to certain persons.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... INCOME TAXES Rules for Computing Credit for Expenses of Work Incentive Programs § 1.50B-5 Limitations... percent of $30,000). If an organization to which section 593 applies is a member of a controlled group (as... estate investment trust is a member of a controlled group (as defined in section 50A (a)(5)), the...

  8. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B sub 1

    SciTech Connect

    Doehmer, J.; Dogra, S.; Friedberg, T.; Monier, S.; Adesnik, M.; Glatt, H.; Oesch, F. )

    1988-08-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltranferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B{sub 1}, which is activated by this enzyme.

  9. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  10. Human adrenodoxin reductase: Two mRNAs encoded by a single gene on chromosome 17cen yields q25 are expressed in steroidogenic tissues

    SciTech Connect

    Solish, S.B.; Picado-Leonard, J.; Morel, Y.; Kuhn, R.W.; Mohandas, T.K.; Hanukoglu, I.; Miller, W.L. )

    1988-10-01

    Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. The authors have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen {yields} q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin.

  11. Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen----q25 are expressed in steroidogenic tissues.

    PubMed

    Solish, S B; Picado-Leonard, J; Morel, Y; Kuhn, R W; Mohandas, T K; Hanukoglu, I; Miller, W L

    1988-10-01

    Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. We have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen----q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin.

  12. Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen----q25 are expressed in steroidogenic tissues.

    PubMed Central

    Solish, S B; Picado-Leonard, J; Morel, Y; Kuhn, R W; Mohandas, T K; Hanukoglu, I; Miller, W L

    1988-01-01

    Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. We have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen----q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin. Images PMID:2845396

  13. Differential Carbon Monoxide Sensitivity of Cytochrome c Oxidase in the Leaves of C3 and C4 Plants 1

    PubMed Central

    Naik, Rajeev M.; Dhage, Ashok R.; Munjal, Shivaji V.; Singh, Prikshayat; Desai, Babasaheb B.; Mehta, Shanti L.; Naik, Manohar S.

    1992-01-01

    CO sensitivity of cytochrome a3 in the leaves of a number of C3 and C4 plants was monitored by the nitrate reductase assay under differing CO to O2 ratios. All the C3 plants were relatively insensitive to CO and required a high CO to O2 ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O2 ratios of 10 or less. On the other hand, the leaves of all C4 plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O2 ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control. PMID:16668775

  14. Differential carbon monoxide sensitivity of cytochrome C oxidase in the leaves of c(3) and c(4) plants.

    PubMed

    Naik, R M; Dhage, A R; Munjal, S V; Singh, P; Desai, B B; Mehta, S L; Naik, M S

    1992-03-01

    CO sensitivity of cytochrome a(3) in the leaves of a number of C(3) and C(4) plants was monitored by the nitrate reductase assay under differing CO to O(2) ratios. All the C(3) plants were relatively insensitive to CO and required a high CO to O(2) ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O(2) ratios of 10 or less. On the other hand, the leaves of all C(4) plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O(2) ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control.

  15. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

    SciTech Connect

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.

  16. Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

    PubMed

    Takaya, N; Suzuki, S; Kuwazaki, S; Shoun, H; Maruo, F; Yamaguchi, M; Takeo, K

    1999-12-15

    Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

  17. Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

    PubMed Central

    2015-01-01

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2–2) for 12-hydroxylation with 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc.108, 7074–7078] and the use of tritium KIE analysis with [12-3H]lauric acid [Northrop, D. B. (1987) Methods Enzymol.87, 607–625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C–H bond-breaking limit the rate of P450 4A11 ω-oxidation. PMID:25203493

  18. Designing a whole-cell biotransformation system in Escherichia coli using cytochrome P450 from Streptomyces peucetius.

    PubMed

    Shrestha, Pramod; Oh, Tae-Jin; Sohng, Jae Kyung

    2008-06-01

    A biotransformation system was designed to co-express CYP107P3 (CSP4), cytochrome P450, from Streptomyces peuceticus, along with CamA (putidaredoxin reductase) and CamB (putidaredoxin) from Pseudomonas putida, the necessary reducing equivalents, in a class I type electron-transfer system in E. coli BL21 (DE3). This was carried out using two plasmids with different selection markers and compatible origins of replication. The study results showed that this biotransformation system was able to mediate the O-dealkylation of 7-ethoxycumarin. PMID:18259876

  19. Mainstream cigarette smoke exposure alters cytochrome P4502G1 expression in F344 rat olfactory mucosa

    SciTech Connect

    Hotchkiss, J.A.; Nikula, K.J.; Lewis, J.L.; Finch, G.L.; Belinsky, S.A.; Dahl, A.R.

    1994-11-01

    Inhalation of mainstream cigarette smoke (MCS) by rats results in multifocal rhinitis, mucous hypersecretion, nasal epithelial hyperplasia and metaplasia, and focal olfactory mucosal atrophy. In humans, cigarette smoking causes long-term, dose-related alterations in olfactory function in both current and former smokers. An olfactory-specific cytochrome P450 has been identified in rabbits and rats. The presence of olfactory-specific P450s, as well as relatively high levels of other biotransformation enzymes, such as NADPH-cytochrome P450 reductase and UDP-glucuronosyl transferase, in the olfactory neuroepithelium suggest that these enzyme systems may play a role in olfaction. This hypothesis is strengthened by the observation that, in rats, the temporal gene activation of P4502G1 coincides with the postnatal increase in the sensitivity of olfactory response to odorants. The purpose of this investigation was to examine the effect of MCS exposure on P4502G1 protein expression.

  20. A world of cytochrome P450s.

    PubMed

    Nelson, David R

    2013-02-19

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.

  1. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

  2. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  3. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

  4. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  5. Synthesis of Nitrate Reductase in Chlorella

    PubMed Central

    Funkhouser, Edward A.; Shen, Teh-Chien; Ackermann, Renate

    1980-01-01

    Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically. PMID:16661310

  6. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    PubMed

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin

    2015-07-01

    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites.

  7. Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c oxidase.

    PubMed

    Pan, L P; Hibdon, S; Liu, R Q; Durham, B; Millett, F

    1993-08-24

    The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to CuA with a rate constant of 1.1 x 10(4) s-1. CuA then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to CuA is greater than 10(5) s-1, and the rate constant for electron transfer from CuA to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c3 family.

    PubMed

    Di Paolo, Roberto E; Pereira, Patrícia M; Gomes, Inês; Valente, Filipa M A; Pereira, Inês A C; Franco, Ricardo

    2006-03-01

    Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c3 family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C-S bonds [nu(C(a)S)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c3 analyzed, namely Type I cytochrome c (3) (TpIc (3)s), Type II cytochrome c (3) (TpIIc (3)s) and Desulfomicrobium cytochromes c3, display different frequency separations for the two nu(C(a)S) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c3. Two larger cytochromes of the cytochrome c3 family display RR spectral characteristics for the nu(C(a)S) lines that are closer to TpIIc3 than to TpIc3. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c3 family display nu(C(a)S) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c3 correlate to differences on the heme-protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.

  9. The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

    PubMed

    ter Beek, Josy; Krause, Nils; Reimann, Joachim; Lachmann, Peter; Ädelroth, Pia

    2013-10-18

    The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

  10. The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection

    PubMed Central

    Shepherd, Mark; Achard, Maud E. S.; Idris, Adi; Totsika, Makrina; Phan, Minh-Duy; Peters, Kate M.; Sarkar, Sohinee; Ribeiro, Cláudia A.; Holyoake, Louise V.; Ladakis, Dimitrios; Ulett, Glen C.; Sweet, Matthew J.; Poole, Robert K.; McEwan, Alastair G.; Schembri, Mark A.

    2016-01-01

    Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract. PMID:27767067

  11. Mammalian cytochromes P-450: Volume I and Volume II

    SciTech Connect

    Guengerich, F.P.

    1987-01-01

    This two volume set summarizes the current knowledge of mammalian cytochromes. Ten chapters cover the current understanding of the enzymology of rat, rabbit, and human liver cytochromes P-450, extrahepatic cytochromes P-450, the diversity of substrates for the individual cytochromes P0-450 proteins, the metabolism of pro-toxicants and -carcinogens by cytochrome P-450, the degradation of cytochrome P-450 proteins, and the regulation of cytochrome P-450 activities in vitro and in vivo. The individual chapters outline the historical development of each area, the approaches which are applied, the current state of knowledge, and future directions towards unresolved questions; and index.

  12. The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

    PubMed

    Díaz-Moreno, Irene; Hulsker, Rinske; Skubak, Pavol; Foerster, Johannes M; Cavazzini, Davide; Finiguerra, Michelina G; Díaz-Quintana, Antonio; Moreno-Beltrán, Blas; Rossi, Gian-Luigi; Ullmann, G Matthias; Pannu, Navraj S; De la Rosa, Miguel A; Ubbink, Marcellus

    2014-08-01

    The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

  13. Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c.

    PubMed

    Elmore, Bradley O; Bergmann, David J; Klotz, Martin G; Hooper, Alan B

    2007-03-01

    Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

  14. The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

    PubMed

    Díaz-Moreno, Irene; Hulsker, Rinske; Skubak, Pavol; Foerster, Johannes M; Cavazzini, Davide; Finiguerra, Michelina G; Díaz-Quintana, Antonio; Moreno-Beltrán, Blas; Rossi, Gian-Luigi; Ullmann, G Matthias; Pannu, Navraj S; De la Rosa, Miguel A; Ubbink, Marcellus

    2014-08-01

    The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process. PMID:24685428

  15. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  16. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae.

    PubMed

    Chang, Qing; Griest, Terry A; Harter, Theresa M; Petrash, J Mark

    2007-03-01

    We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  17. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  18. Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

    PubMed

    Zaman, Khalequz; Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J; Lewis, Stephen J; Gaston, Benjamin

    2016-02-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

  19. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease.

    PubMed

    Nicoli, Elena-Raluca; Al Eisa, Nada; Cluzeau, Celine V M; Wassif, Christopher A; Gray, James; Burkert, Kathryn R; Smith, David A; Morris, Lauren; Cologna, Stephanie M; Peer, Cody J; Sissung, Tristan M; Uscatu, Constantin-Daniel; Figg, William D; Pavan, William J; Vite, Charles H; Porter, Forbes D; Platt, Frances M

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  20. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  1. Cummins Engine Company B5.9 Propane Engine Development, Certification, and Demonstration Project

    SciTech Connect

    The ADEPT Group, Inc.

    1998-12-18

    The objective of this project was to successfuly develop and certify an LPG-dedicated medium-duty original equipment manufacturer (OEM) engine that could be put into production. The engine was launched into production in 1994, and more than 800 B5.9G engines are now in service in the United States and abroad. This engine is now offered by more than 30 bus and truck OEMs.

  2. The standards process: Technical committee X3B5 digital magnetic tape

    NASA Technical Reports Server (NTRS)

    Cheatham, Sam

    1993-01-01

    The definition of X3B5, where it fits in the national and international standards development process, and how it interfaces and influences the world community of standards developers are provided. Details concerning the focus of the committee, how it operates, and what the group sees as the future trends in the area of interchange standards utilizing the multifaceted, ubiquitous magnetic tape are presented.

  3. O(3P) attack on boranes. II. B5H9

    NASA Astrophysics Data System (ADS)

    Cheng, H.-Z.; Bauer, S. H.

    1991-06-01

    When B5H9 is injected into a stream of He that is carrying O(3P) atoms (approximately 100/1), at a total pressure of 5-15 Torr, a blue-green flame develops. The major chemiluminescent species is BO(A 2Π). While its translational and rotational temperatures are ≊350 K, the vibrational temperature in the A state is high, ≊3800 K. From among the many products of this reaction, the OH radical can be most easily quantitated by measuring the intensity of its laser-induced fluorescence. The central streamline from a flow-tube reactor was extracted into an evacuated plenum via a pinhole. The time-intensity profile was calibrated using C2H6 for the fuel. Check runs were made with B2H6. A multistep mechanism was developed for B5H9+O(3P) that simulates the shape as well as the magnitude of the OH concentration over a reactor residence time 0.5-10 ms. Less than a dozen crucial reactions were identified by means of an extended sensitivity analysis. Breakdown schemes for the oxidation of B2H6 and B5H9 have been developed.

  4. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery. PMID:16859410

  5. Islet secretory granules contain cytochrome b561.

    PubMed

    Mackin, R B; Jones, D P; Noe, B D

    1986-08-01

    A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones. PMID:3525285

  6. Cytochrome bd Displays Significant Quinol Peroxidase Activity

    PubMed Central

    Al-Attar, Sinan; Yu, Yuanjie; Pinkse, Martijn; Hoeser, Jo; Friedrich, Thorsten; Bald, Dirk; de Vries, Simon

    2016-01-01

    Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme. PMID:27279363

  7. Regulation of nitrate reductase (NR) synthesis investigated by using mutants of Chl. sorokiniana partially NR deficient

    SciTech Connect

    Knobloch, O.; Tischner, R.

    1986-04-01

    After X-ray irradiation 13 NR and 8 nitrite reductase (NiR) deficient mutants of Chl.sorokiniana were obtained. In order to assure best experimental conditions for the characterization of the NR mutants, for which NO/sub 3//sup -/-containing medium in fact is a N-medium, they transferred wild type cells from NH/sub 4//sup +/ to NO/sub 3//sup -/ or N-medium, respectively. It turned out, that NO/sub 3//sup -/ is not necessary for starting de-novo-synthesis of NR. Therefore NR in Chlorella is a derepressible enzyme rather than an inducible one. Maximum amount of NR is present 80 min. after transfer of cells. Derepression experiments with the mutant strains characterized 3 of them as defect in Mo-co subunit of NR with best cytochrome c reductase (CCR)-activity, although xanthine oxidase (XO) was inducible. One other mutant is CCR-defect but contains intact Mo-co. The latter strain produced 4-6 times more Mo-co than the wild type, giving some evidence for an unbalanced self-regulation of NR-synthesis. Another strain lacked XO-activity, indicating a common cofactor among XO and NR as reported for other organisms.

  8. Human Heme Oxygenase-1 Efficiently Catabolizes Heme in the Absence of Biliverdin Reductase

    PubMed Central

    Huber, Warren J.; Backes, Wayne L.

    2010-01-01

    Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR. PMID:20679134

  9. Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin- and a peroxidase-like heme-protein.

    PubMed

    Ascenzi, Paolo; Coletta, Massimo; Wilson, Michael T; Fiorucci, Laura; Marino, Maria; Polticelli, Fabio; Sinibaldi, Federica; Santucci, Roberto

    2015-02-01

    Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc.

  10. Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin- and a peroxidase-like heme-protein.

    PubMed

    Ascenzi, Paolo; Coletta, Massimo; Wilson, Michael T; Fiorucci, Laura; Marino, Maria; Polticelli, Fabio; Sinibaldi, Federica; Santucci, Roberto

    2015-02-01

    Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc. PMID:25857294

  11. The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective

    PubMed Central

    Ducluzeau, Anne-Lise; Schoepp-Cothenet, Barbara; van Lis, Robert; Baymann, Frauke; Russell, Michael J.; Nitschke, Wolfgang

    2014-01-01

    Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes. PMID:24968694

  12. The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective.

    PubMed

    Ducluzeau, Anne-Lise; Schoepp-Cothenet, Barbara; van Lis, Robert; Baymann, Frauke; Russell, Michael J; Nitschke, Wolfgang

    2014-09-01

    Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes.

  13. miR-487b-5p Regulates Temozolomide Resistance of Lung Cancer Cells Through LAMP2-Medicated Autophagy.

    PubMed

    Bao, Liang; Lv, Lei; Feng, Jinping; Chen, Yuyu; Wang, Xinhua; Han, Shuguang; Zhao, Hongqing

    2016-08-01

    Temozolomide (TMZ) is a standard agent used in the treatment of various types of cancers, including lung carcinoma, but TMZ resistance is common and accounts for many treatment failures. We investigated miRNA-487b-5p (miR-487b-5p) was highly expressed in A549 and H1299 cells which acquired TMZ resistance. Suppression of miR-487b-5p had overt effects on cellular proliferation and migration in the presence of TMZ. On the other hand, knockdown of miR-487b-5p resulted in increased survival and moderate tumor growth in vivo. In addition, the decreased cellular proliferation following miR-487b-5p suppression was linked to enhanced autophagy, evident by drastically increased levels of LC3-II, BECLIN1, and LAMP2 when miR-487b-5p was knocked down. Further analysis revealed that LAMP2 might be the target gene of miR-487b-5p. In conclusion, our study suggested that miR-487b-5p may be a potential biomarker of acquired TMZ resistance in lung cancer cells, and miR-487b-5p inhibition can be further explored as a chemotherapy target in the treatment of TMZ-resistant lung carcinoma.

  14. Reduction of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by oxygen sensitive nitro reductase enzymes

    SciTech Connect

    Shah, M.M.; Spain, J.C.

    1995-12-01

    Reduction of nitroaromatic compounds by nitroreductase enzymes generally leads to the formation of the corresponding amines. However, we recently found that the incubation of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) with ferredoxin-NADP oxidoreductase, an oxygen sensitive nitroreductase from spinach in the presence of NADPH led to the elimination of the nitramine nitro group from tetryl and the formation of N-methylpicramide (NMP). Other oxygen sensitive nitroreductase enzymes including glutathione reductase, xanthine oxidase, and cytochrome c reductase were also able to release nitrite from tetryl. Nitrite was not eliminated from tetryl by an oxygen insensitive nitrobenzene reductase. For every mole of tetryl reduced, one mole each of nitrite and NMP were produced. The rate of nitrite elimination was inhibited under aerobic conditions. Subsequent oxygen uptake studies suggested that under aerobic conditions, molecular oxygen was reduced by FNR and tetryl served as the redox mediator. Our results suggest that under aerobic conditions; tetryl is reduced to the nitroanion radical by the enzyme and this radical is involved in the reduction of molecular oxygen.

  15. Structure of aldose reductase from Giardia lamblia

    PubMed Central

    Ferrell, M.; Abendroth, J.; Zhang, Y.; Sankaran, B.; Edwards, T. E.; Staker, B. L.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior. PMID:21904059

  16. Steroid 5α-reductase 2 deficiency.

    PubMed

    Mendonca, Berenice B; Batista, Rafael Loch; Domenice, Sorahia; Costa, Elaine M F; Arnhold, Ivo J P; Russell, David W; Wilson, Jean D

    2016-10-01

    Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex. PMID:27224879

  17. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome.

  18. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. PMID:25091281

  19. Discovery of pinoresinol reductase genes in sphingomonads.

    PubMed

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  20. Thermal stability of the polyheme cytochrome c3 superfamily.

    PubMed

    Florens, L; Bianco, P; Haladjian, J; Bruschi, M; Protasevich, I; Makarov, A

    1995-10-16

    The cytochrome c3 superfamily includes Desulfovibrio polyheme cytochromes c. We report the characteristic thermal stability parameters of the Desulfovibrio desulfuricans Norway (D.d.N.) cytochromes c3 (M(r) 13,000 and M(r) 26,000) and the Desulfovibrio vulgaris Hildenborough (D.v.H.) cytochrome c3 (M(r) 13,000) and high molecular mass cytochrome c (Hmc), as obtained with the help of electronic spectroscopy, voltammetric techniques and differential scanning calorimetry. The polyheme cytochromes are denatured over a wide range of temperatures: the D.v.H. cytochrome c3 is highly thermostable (Td = 121 degrees C) contrary to the D.d.N. protein (Td = 73 degrees C). The thermostability of the polyheme cytochromes is redox state dependent. The results are discussed in the light of the structural and functional relationships within the cytochrome c3 superfamily. PMID:7589483

  1. Comparison of Monte Carlo simulations of cytochrome b6f with experiment using Latin hypercube sampling.

    PubMed

    Schumaker, Mark F; Kramer, David M

    2011-09-01

    We have programmed a Monte Carlo simulation of the Q-cycle model of electron transport in cytochrome b(6)f complex, an enzyme in the photosynthetic pathway that converts sunlight into biologically useful forms of chemical energy. Results were compared with published experiments of Kramer and Crofts (Biochim. Biophys. Acta 1183:72-84, 1993). Rates for the simulation were optimized by constructing large numbers of parameter sets using Latin hypercube sampling and selecting those that gave the minimum mean square deviation from experiment. Multiple copies of the simulation program were run in parallel on a Beowulf cluster. We found that Latin hypercube sampling works well as a method for approximately optimizing very noisy objective functions of 15 or 22 variables. Further, the simplified Q-cycle model can reproduce experimental results in the presence or absence of a quinone reductase (Q(i)) site inhibitor without invoking ad hoc side-reactions.

  2. Comparison of Monte Carlo simulations of cytochrome b6f with experiment using Latin hypercube sampling.

    PubMed

    Schumaker, Mark F; Kramer, David M

    2011-09-01

    We have programmed a Monte Carlo simulation of the Q-cycle model of electron transport in cytochrome b(6)f complex, an enzyme in the photosynthetic pathway that converts sunlight into biologically useful forms of chemical energy. Results were compared with published experiments of Kramer and Crofts (Biochim. Biophys. Acta 1183:72-84, 1993). Rates for the simulation were optimized by constructing large numbers of parameter sets using Latin hypercube sampling and selecting those that gave the minimum mean square deviation from experiment. Multiple copies of the simulation program were run in parallel on a Beowulf cluster. We found that Latin hypercube sampling works well as a method for approximately optimizing very noisy objective functions of 15 or 22 variables. Further, the simplified Q-cycle model can reproduce experimental results in the presence or absence of a quinone reductase (Q(i)) site inhibitor without invoking ad hoc side-reactions. PMID:21221830

  3. Influence of P ion on Sr2B5O9Cl:Eu for TL dosimetry

    NASA Astrophysics Data System (ADS)

    Oza, Abha H.; Dhoble, N. S.; Dhoble, S. J.

    2015-02-01

    This paper investigates luminescence properties of Sr2B5O9Cl:Eu phosphor prepared by modified solid state diffusion. The influence of Phosphorous ion as codopant is also explained in detail. The structural confirmation of the sample was done using the XRD technique. SEM revealed the microcrystalline nature of the prepared phosphor. The characteristic Eu2+ emission at 437 nm and 423 nm was observed for Sr2B5O9Cl:Eu and Sr2B5O9Cl:P,Eu, respectively under 338 nm excitation. Samples in powder form were irradiated with different doses under γ-ray irradiation with 60Co source and the TL glow curves for both Sr2B5O9Cl:Eu and Sr2B5O9Cl:P,Eu samples were studied. In case of Sr2B5O9Cl:Eu phosphor, single glow curve nature centered on 260 °C with a shoulder peak around 144 °C was observed. However; Sr2B5O9Cl:P,Eu have shown slight different and broad glow curve nature. The TL sensitivity in both the cases was compared with CaSO4:Dy phosphor. Sr2B5O9Cl:Eu sample have shown 1.17 times less sensitivity than CaSO4:Dy and for Sr2B5O9Cl:P,Eu it was found to be equal to CaSO4:Dy and Sr2B5O9Cl:P,Eu is 1.21 times more sensitive than Sr2B5O9Cl:Eu. Other TL properties like dose response, fading and reusability were studied for both the samples. The trapping parameters for both the samples were calculated using computerized glow curve deconvolution and reported in this paper.

  4. Biochemical Characterization of a Haloalkane Dehalogenase DadB from Alcanivorax dieselolei B-5

    PubMed Central

    Li, Anzhang; Shao, Zongze

    2014-01-01

    Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest kcat/Km value toward 1,3-dibromopropane (Km = 0.82 mM, kcat/Km = 16.43 mM−1·s−1). DadB aggregates fast in the buffers with pH≤7.0, while keeps stable in monomer form when pH≥7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications. PMID:24586552

  5. Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors.

    PubMed

    Bücking, Clemens; Popp, Felix; Kerzenmacher, Sven; Gescher, Johannes

    2010-05-01

    The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.

  6. Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c1.

    PubMed

    Heacock, D H; Liu, R Q; Yu, C A; Yu, L; Durham, B; Millett, F

    1993-12-25

    The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a 0.4-microseconds laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 nM and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mM ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

  7. IAU Resolution 2009 B5 - Commission 50 Draft Action Plan - Presentation and Discussion

    NASA Astrophysics Data System (ADS)

    Green, R. F.

    2015-03-01

    IAU Resolution 2009 B5 calls on IAU members to protect the public's right to an unpolluted night sky as well as the astronomical quality of the sky around major research observatories. The multi-pronged approach of Commission 50 includes working with the lighting industry for appropriate products from the solid state revolution, arming astronomers with training and materials for presentation, selective endorsement of key protection issues, cooperation with several other IAU commissions for education and outreach, and provision of clear quantitative priorities for outdoor lighting standards.

  8. Dynamic apparent transition resistance data in spot welding of aluminized 22MnB5.

    PubMed

    Kaars, Jonny; Mayr, Peter; Koppe, Kurt

    2016-09-01

    In-situ resistance measurements of aluminized 22MnB5 steel using a current ramp of 500 A/ms at welding force levels from 2 kN to 8 kN were conducted to obtain data on the dynamic resistance behaviour in spot welding of the material for varying mechanical and electrical loads. The data has been successfully used to calibrate a numerical transition resistance model (KMK-model, Kaars et al., 2016 [1]) in Kaars et al. (2016) [2].

  9. Dynamic apparent transition resistance data in spot welding of aluminized 22MnB5.

    PubMed

    Kaars, Jonny; Mayr, Peter; Koppe, Kurt

    2016-09-01

    In-situ resistance measurements of aluminized 22MnB5 steel using a current ramp of 500 A/ms at welding force levels from 2 kN to 8 kN were conducted to obtain data on the dynamic resistance behaviour in spot welding of the material for varying mechanical and electrical loads. The data has been successfully used to calibrate a numerical transition resistance model (KMK-model, Kaars et al., 2016 [1]) in Kaars et al. (2016) [2]. PMID:27547795

  10. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase.

    PubMed

    Wei, Yifeng; Li, Bin; Prakash, Divya; Ferry, James G; Elliott, Sean J; Stubbe, JoAnne

    2015-12-01

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.

  11. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

  12. Label-free photoacoustic microscopy of cytochromes.

    PubMed

    Zhang, Chi; Zhang, Yu Shrike; Yao, Da-Kang; Xia, Younan; Wang, Lihong V

    2013-02-01

    Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (~420 nm), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.

  13. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  14. Cytochrome P450 monooxygenase system in echinoderms.

    PubMed

    den Besten, P J

    1998-11-01

    The results of a limited number of studies on echinoderms provide evidence for the presence of a cytochrome P450 monooxygenase system in representatives of three classes of the phylum Echinodermata: the asteroids (sea stars), holothuroids (sea cucumbers) and echinoids (sea urchins). The monooxygenase system has been demonstrated to be involved in the metabolism of xenobiotic compounds, but is assumed to have its primary function in the metabolism of endogenous substrates, such as steroids. Available data on P450 cofactor requirement, P450-dependent metabolism of benzo[a]pyrene, studies with classical inhibitors of P450, specificity of P450 induction by planar compounds, and the changes in the benzo[a]pyrene metabolite profile in induced animals suggest similarities with the MO system present in vertebrates. However, the relatively high capacity of the monooxygenase system in sea stars to catalyse reactions with organic hydroperoxide as donor for activated oxygen, and the low induceability during exposure to xenobiotics indicate also important differences between the echinoderm cytochrome P450 monooxygenase system and that of vertebrates. Some evidence was found for the existence of different forms of cytochrome P450 in sea stars. Catalytic functions of the cytochrome P450 monooxygenase system of sea stars in the metabolism of steroids may be suppressed as a result of the induction of cytochrome P450 by xenobiotics. PMID:9972455

  15. Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

    PubMed Central

    Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu

    2013-01-01

    Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM−1 s−1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

  16. Quorum-sensing-directed protein expression in Serratia proteamaculans B5a.

    PubMed

    Christensen, Allan B; Riedel, Kathrin; Eberl, Leo; Flodgaard, Lars R; Molin, Søren; Gram, Lone; Givskov, Michael

    2003-02-01

    N-Acyl-L-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

  17. Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions.

    PubMed

    Doceul, Virginie; Hollinshead, Michael; Breiman, Adrien; Laval, Kathlyn; Smith, Geoffrey L

    2012-09-01

    Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

  18. Ab initio study on structural, electronic properties, and hardness of re-doped W2B5

    NASA Astrophysics Data System (ADS)

    Feng, ShiQuan; Li, XiaoDong; Su, Lei; Li, HaiNing; Yang, Hongyan; Cheng, Xinlu

    2016-11-01

    Using first principle calculations method, we calculated the structural stability, electronic properties and hardness for three Re-doped hexagonal W2B5 compounds. The electronic properties were carried out to discuss the effect of doped Re on the conductivity, chemical bonding components and orbital hybridization of W2B5. What is more, the hardness of these three doped crystals was calculated by a semiempirical method considering the role of metallic components. A Re-doped W2B5 compound with a greater hardness value than that of pure W2B5 was obtained. And the effect of doping on the hardness of hexagonal W2B5was discussed.

  19. Periplasmic Cytochrome c(3) of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

    SciTech Connect

    Elias, Dwayne A.; Suflita, Joseph M.; McInerney, Michael J.; Krumholz, Lee R.

    2004-01-01

    Kinetic parameters and the role of cytochrome c3 in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (Km 220 uM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H2 and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H2 and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H2, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate or pyruvate-reduced, but not H2-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H2 was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H2 was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c3 is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.

  20. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

    PubMed Central

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-01-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies. PMID:23844938

  1. Evidence for the involvement of two heterodisulfide reductases in the energy-conserving system of Methanomassiliicoccus luminyensis.

    PubMed

    Kröninger, Lena; Berger, Stefanie; Welte, Cornelia; Deppenmeier, Uwe

    2016-02-01

    Methanomassiliicoccus luminyensis was isolated from the human gut, and requires H2 and methanol or methylamines to produce methane. The organism lacks cytochromes, indicating that it cannot couple membrane-bound electron transfer reactions with extrusion of H(+) or Na(+) ions using known methanogenic pathways. Furthermore, M. luminyensis contains a soluble hydrogenase/heterodisulfide reductase complex (MvhAGD/HdrABC) as found in obligate hydrogenotrophic methanogens, but the energy-conserving methyltransferase (MtrA-H) is absent. Thus, the question arises as to how this species synthesizes ATP. We present evidence that M. luminyensis uses two types of heterodisulfide reductases (HdrABC and HdrD) in a novel process for energy conservation. Quantitative RT-PCR studies revealed that genes encoding these heterodisulfide reductases showed high expression levels. Other genes with high transcript abundance were fpoA (part of the operon encoding the 'headless' F420 H2 dehydrogenase) and atpB (part of the operon encoding the A1 Ao ATP synthase). High activities of the soluble heterodisulfide reductase HdrABC and the hydrogenase MvhADG were found in the cytoplasm of M. luminyensis. Also, heterologously produced HdrD was able to reduce CoM-S-S-CoB using reduced methylviologen as an electron donor. We propose that membrane-bound electron transfer is based on conversion of two molecules of methanol and concurrent formation of two molecules of the heterodisulfide CoM-S-S-CoB. First the HdrABC/MvhADG complex catalyzes the H2 -dependent reduction of CoM-S-S-CoB and formation of reduced ferredoxin. In a second cycle, reduced ferredoxin is oxidized by the 'headless' F420 H2 dehydrogenase, thereby translocating up to 4 H(+) across the membrane, and electrons are channeled to HdrD for reduction of the second heterodisulfide.

  2. A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes.

    PubMed

    De Mot, René; Parret, Annabel H A

    2002-11-01

    The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications. PMID:12419614

  3. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    PubMed Central

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S. P.; Wade, Rebecca C.

    2011-01-01

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix. PMID:21852944

  4. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.; Wade, Rebecca C.

    2011-08-11

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

  5. Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes

    PubMed Central

    Osuda, Yukiho; Shinzawa-Itoh, Kyoko; Tani, Kazutoshi; Maeda, Shintaro; Yoshikawa, Shinya; Tsukihara, Tomitake; Gerle, Christoph

    2016-01-01

    Mitochondrial cytochrome c oxidase utilizes electrons provided by cytochrome c for the active vectorial transport of protons across the inner mitochondrial membrane through the reduction of molecular oxygen to water. Direct structural evidence on the transient cytochrome c oxidase–cytochrome c complex thus far, however, remains elusive and its physiological relevant oligomeric form is unclear. Here, we report on the 2D crystallization of monomeric bovine cytochrome c oxidase with tightly bound cytochrome c at a molar ratio of 1:1 in reconstituted lipid membranes at the basic pH of 8.5 and low ionic strength. PMID:26754561

  6. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  7. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    PubMed Central

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B.

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

  8. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System.

    PubMed

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H - referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner - diflavin reductase - by fusing both enzymes individually to the hydrophobin HFBI - a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

  9. Genetic characterization of Bagarius species using cytochrome c oxidase I and cytochrome b genes.

    PubMed

    Nagarajan, Muniyandi; Raja, Manikam; Vikram, Potnuru

    2016-09-01

    In this study, we first inferred the genetic variability of two Bagarius bagarius populations collected from Ganges and Brahmaputra rivers of India using two mtDNA markers. Sequence analysis of COI gene did not show significant differences between two populations whereas cytochrome b gene showed significant differences between two populations. Followed by, genetic relationship of B. bagarius and B. yarrielli was analyzed using COI and cytochrome b gene and the results showed a higher level genetic variation between two species. The present study provides support for the suitability of COI and cytochrome b genes for the identification of B. bagarius and B. yarrielli.

  10. Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation.

    PubMed

    Mayhew, M P; Reipa, V; Holden, M J; Vilker, V L

    2000-01-01

    Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system. PMID:10933836

  11. Regulation and function of cytochrome c' in Rhodobacter sphaeroides 2.4.3.

    PubMed

    Choi, Peter S; Grigoryants, Vladimir M; Abruña, Hector D; Scholes, Charles P; Shapleigh, James P

    2005-06-01

    Cytochrome c' (Cyt c') is a c-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in Rhodobacter sphaeroides 2.4.3, designated cycP, was isolated and sequenced. Northern blot analysis and beta-galactosidase assays demonstrated that cycP transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another Rhodobacter species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt c' levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt c' under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt c' binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt c' has NO reductase activity. Taken together, these results suggest a model where Cyt c' in R. sphaeroides 2.4.3 may shuttle NO to the membrane, where it can be reduced.

  12. Crp-dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis.

    PubMed

    Fu, Huihui; Chen, Haijiang; Wang, Jixuan; Zhou, Guangqi; Zhang, Haiyan; Zhang, Lili; Gao, Haichun

    2013-08-01

    Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (cyclic AMP receptor protein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp-independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.

  13. AS2077715 is a selective inhibitor of fungal mitochondrial cytochrome bc₁ complex.

    PubMed

    Ohsumi, Keisuke; Watanabe, Masato; Fujie, Akihiko

    2014-10-01

    AS2077715 is a novel antifungal metabolite produced by the newly isolated fungal strain Capnodium sp. 339855. This compound has an analogous structure to funiculosin, an inhibitor of mitochondrial cytochrome bc1 complex (complex III). AS2077715 inhibited ubiquinol-cytochrome c reductase activity of Trichophyton mentagrophytes complex III with an IC50 of 0.9 ng ml(-1), while 6000-20,000 ng ml(-1) AS2077715 was required to obtain comparable inhibition of mammalian complex III. This inhibitor also suppressed the growth of T. mentagrophytes with a MIC of 0.08 μg ml(-1), while cytotoxicity for mammalian cells was >6 μg ml(-1). These results indicate that AS2077715 is a selective inhibitor of fungal mitochondrial complex III. AS2077715 in doses of 1 μg ml(-1) or greater showed fungicidal activity against T. mentagrophytes within 2 h of incubation. This early-onset effect of fungicidal activity was also exhibited by other complex III inhibitors. These results suggest that inhibition of complex III is a promising strategy for designing anti-Trichophyton agents and that AS2077715 can be a potential drug candidate for treating Trichophyton infections.

  14. Hydroxylation of phenol to catechol by Candida tropicalis: involvement of cytochrome P450.

    PubMed

    Stiborová, M; Suchá, V; Miksanová, M; Páca, J; Páca, J

    2003-06-01

    Microsomal preparations isolated from yeast Candida tropicalis (C. tropicalis) grown on three different media with or without phenol were isolated and characterized for the content of cytochrome P450 (CYP) (EC 1.14.15.1). While no CYP was detected in microsomes of C. tropicalis grown on glucose as the carbon source, evidence was obtained for the presence of the enzyme in the microsomes of C. tropicalis grown on media containing phenol. Furthermore, the activity of NADPH: CYP reductase, another enzyme of the microsomal CYP-dependent system, was markedly higher in cells grown on phenol. Microsomes of these cells oxidized phenol. The major metabolite formed from phenol by microsomes of C. tropicalis was characterized by UV/vis absorbance and mass spectroscopy as well as by the chromatographic properties on HPLC. The characteristics are identical to those of catechol. The formation of catechol was inhibited by CO, the inhibitor of CYP, and correlated with the content of cytochrome P450 in microsomes. These results, the first report showing the ring hydroxylation of phenol to catechol with the microsomal enzyme system of C. tropicalis, strongly suggest that CYP-catalyzed reactions are responsible for this hydroxylation. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by the C. tropicalis strain.

  15. The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450.

    PubMed

    Stresser, D M; Bjeldanes, L F; Bailey, G S; Williams, D E

    1995-08-01

    Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now report a similar potent inhibition by 3,3'-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3'-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans.

  16. Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

    PubMed

    Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

    2006-05-01

    Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

  17. Radical scavengers as ribonucleotide reductase inhibitors.

    PubMed

    Basu, Arijit; Sinha, Barij Nayan

    2012-01-01

    This paper compiled all the previous reports on radical scavengers, an interesting class of ribonucleotide reductase inhibitors. We have highlighted three key research areas: chemical classification of radical scavengers, structural and functional aspects of the radical site, and progress in drug designing for radical scavengers. Under the chemical classification section, we have recorded the discovery of hydroxyurea followed by discussions on hydroxamic acids, amidoximes, hydroxyguanidines, and phenolic compounds. In the next section, we have compiled the structural information for the radical site obtained from different crystallographic and theoretical studies. Finally, we have included the reported ligand based and structure based drug-designing studies.

  18. [Degradation of Steroidal Hormones by Salt-tolerant Altererythrobacter Strain MH-B5: Degradation Characteristics, Metabolites and Its Immobilization].

    PubMed

    Ma, Cong; Qin, Dan; Sun, Qian; Yu, Chang-ping

    2015-12-01

    A steroidal hormone degrading bacterium, named MH-B5, was isolated from saline lagoon. Several steroidal hormones including estrone, 17-β-estradiol, estriol and testosterone could be utilized as sole carbon source by strain MH-B5. Analysis of 16S rRNA gene sequence showed that it belonged to genus Altererythrobacter. Growth of strain MH-B5 was observed at salinities of 0-7% and at pH of 5.5-9.0. The main degradation metabolites of testosterone by strain MH-B5 were identified as 4-androstenedione, 1-dehydrotestosterone and androstadienedione using ultra performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight mass spectrometry (Q-TOF MS). Mono-carrier and multi-carrier cell immobilization techniques were successfully applied to this steroid-degrading bacterium. Batch estrogen degradation study showed that immobilized strain MH-B5 could effectively degrade 2 mg · L⁻¹ of 17β-estradiol and its metabolite estrone. The results mentioned above demonstrated the capability of MH-B5 to degrade both estrogens and testosterone and the potential application of using immobilized MH-B5 cells in bioremediation of steroidal hormone pollution in saline environment. PMID:27012005

  19. Coxsackievirus B5 induced apoptosis of HeLa cells: Effects on p53 and SUMO

    SciTech Connect

    Gomes, Rogerio; Guerra-Sa, Renata; Arruda, Eurico

    2010-01-20

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  20. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes.

    PubMed

    Reed, James R; Cawley, George F; Ardoin, Taylor G; Dellinger, Barry; Lomnicki, Slawomir M; Hasan, Farhana; Kiruri, Lucy W; Backes, Wayne L

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition.

  1. Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.

    PubMed

    Zhao, Fanglong; Bai, Peng; Liu, Ting; Li, Dashuai; Zhang, Xiangmei; Lu, Wenyu; Yuan, Yingjin

    2016-08-01

    Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc. PMID:26757342

  2. B5r gene based sequence analysis of Indian buffalopox virus isolates in relation to other orthopoxviruses.

    PubMed

    Singh, R K; Balamurugan, V; Hosamani, M; DE, U K; Chandra, B M; Krishnappa M P, G

    2007-01-01

    We determined complete nucleotide sequence of B5R gene homologue of Vaccinia virus (VACV) in five Buffalopox virus (BPXV) isolates of Indian origin. The obtained sequences were compared with themselves and with corresponding sequences of the other orthopoxviruses. Sequence analysis revealed 99.799.8% and 99.499.7% identities among the BPXV isolates for B5R gene at the nucleotide and amino acid levels, respectively. Sequence identities of B5R gene between BPXV and VACV isolates (98.199.7%) or other orthopoxviruses (95.699.2%) showed highly conserved nature of this protein and a closer relationship of BPXV isolates to VACV than to other orthopoxviruses.

  3. Cytochrome C: A Biochemistry Laboratory Course

    ERIC Educational Resources Information Center

    Vincent, John B.; Woski, Stephen A.

    2005-01-01

    A laboratory course called cytochrome c that focuses on the theme of biochemical research is presented. The students follow this course by incorporating team-investigation and self-directed experimentation that provides them an opportunity to experience the excitement of research.

  4. Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants.

    PubMed Central

    Werck-Reichhart, D; Gabriac, B; Teutsch, H; Durst, F

    1990-01-01

    The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide. PMID:2241905

  5. Importance of cytochromes in cyclization reactions: quantum chemical study on a model reaction of proguanil to cycloguanil.

    PubMed

    Arfeen, Minhajul; Patel, Dhilon S; Abbat, Sheenu; Taxak, Nikhil; Bharatam, Prasad V

    2014-10-30

    Proguanil, an anti-malarial prodrug, undergoes