Other search tools

About this data

The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.

If you have any questions or comments, please contact us.

Results: 1 to 10 of 63

Publication Record

Connections

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.
Gonzalez E, Guengerich FP
(2017) J Biol Chem 292: 13168-13185
MeSH Terms: 17-alpha-Hydroxypregnenolone, Androstenedione, Animals, Binding Sites, Biocatalysis, Cytochrome P-450 Enzyme Inhibitors, Cytochromes b5, Dehydroepiandrosterone, Humans, Imidazoles, Kinetics, Ligands, Models, Molecular, NADPH-Ferrihemoprotein Reductase, Naphthalenes, Oxidation-Reduction, Pregnenolone, Progesterone, Protein Conformation, Rats, Recombinant Proteins, Stereoisomerism, Steroid 17-alpha-Hydroxylase
Show Abstract · Added March 14, 2018
Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome (), but the exact role of in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. did not enhance reaction rates by decreasing the rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. ()-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC values for ()-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
0 Communities
1 Members
0 Resources
23 MeSH Terms
Cytochrome P450 2S1 is reduced by NADPH-cytochrome P450 reductase.
Xiao Y, Shinkyo R, Guengerich FP
(2011) Drug Metab Dispos 39: 944-6
MeSH Terms: Aerobiosis, Anaerobiosis, Animals, Anthraquinones, Cytochrome P-450 Enzyme System, Electron Transport, Escherichia coli, Humans, NADPH-Ferrihemoprotein Reductase, Oxidation-Reduction, Oxygen, Rats
Show Abstract · Added March 7, 2014
Cytochrome P450 (P450) 2S1 is one of the orphan P450s without a clear physiological function. Controversy has arisen as to whether it can interact with NADPH-P450 reductase and accept electrons. The reduction of 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (AQ4N) by P450 2S1 was confirmed, and the NADPH consumption rates were measured aerobically and anaerobically in the absence and presence of the drug. The reduction kinetics of P450 2S1 were rapid, as measured by stopped-flow kinetics. These results confirm that P450 2S1 can be reduced by NADPH-P450 reductase and suggest normal mixed-function oxidase roles of P450 2S1 to be revealed.
0 Communities
1 Members
0 Resources
12 MeSH Terms
Clinical, genetic, and enzymatic characterization of P450 oxidoreductase deficiency in four patients.
Sahakitrungruang T, Huang N, Tee MK, Agrawal V, Russell WE, Crock P, Murphy N, Migeon CJ, Miller WL
(2009) J Clin Endocrinol Metab 94: 4992-5000
MeSH Terms: Adolescent, Adult, Bone and Bones, Catalysis, Cytochromes c, DNA, Disorders of Sex Development, Escherichia coli, Female, Genetic Vectors, Genitalia, Hormones, Humans, Infant, Newborn, Infertility, Male, Mutation, NADP, NADPH-Ferrihemoprotein Reductase, Pregnancy, Steroid 17-alpha-Hydroxylase, Syndrome
Show Abstract · Added May 5, 2016
CONTEXT - P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility.
OBJECTIVE - We describe four patients with POR deficiency and identify and characterize the activities of their mutations. A 46,XY male with micropenis and two 46,XX female infants with genital ambiguity presented with skeletal malformations, and a 46,XX adolescent presented with primary amenorrhea, elevated 17alpha-hydroxyprogesterone, and low sex steroids.
METHODS - The coding regions of the POR gene were sequenced, and the identified mutations were recreated in human POR cDNA expression vectors lacking 27 N-terminal residues. POR and human P450c17 were expressed in bacteria. POR activity was measured by four assays: reduction of cytochrome c, oxidation of reduced nicotinamide adenine dinucleotide phosphate, and support of the 17alpha-hydroxylase and 17,20 lyase activities of P450c17.
RESULTS - All four patients were compound heterozygotes for POR mutations, including five novel mutations: L577R, N185K, delE217, and frameshift mutations 1363delC and 697-698insGAAC. N185K and delE217 lacked measurable activity in the assays based on P450c17 but retained partial activity in the assays based on cytochrome c. As assessed by V(max)/Km, L577R supported 46% of 17alpha-hydroxylase activity but only 27% of 17,20 lyase activity. Computational modeling of these novel mutants revealed the structural basis for their reduced or absent activities.
CONCLUSION - These patients illustrate the broad clinical spectrum of POR deficiency, including amenorrhea and infertility as the sole manifestation. POR assays based on P450c17 correlate well with hormonal and clinical phenotypes.
0 Communities
1 Members
0 Resources
22 MeSH Terms
Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase.
Guengerich FP, Martin MV, Sohl CD, Cheng Q
(2009) Nat Protoc 4: 1245-51
MeSH Terms: Animals, Cytochrome P-450 Enzyme System, Humans, Microsomes, Liver, NADPH-Ferrihemoprotein Reductase, Rats, Spectrophotometry
Show Abstract · Added March 26, 2014
Cytochrome P450 (P450) enzymes are important in the metabolism of steroids, vitamins, carcinogens, drugs and other compounds. Two of the commonly used assays in this field are the measurements of total P450 and NADPH-P450 reductase in biological preparations. A detailed protocol is presented for the measurement of P450 by its spectral properties, along with a protocol for measuring NADPH-P450 reductase by its NADPH-cytochrome c reduction activity. Each assay can be completed in 5-10 min. Detailed explanations for the rationale of particular sequences in the protocols are provided, along with potential confounding problems.
0 Communities
1 Members
0 Resources
7 MeSH Terms
A second FMN binding site in yeast NADPH-cytochrome P450 reductase suggests a mechanism of electron transfer by diflavin reductases.
Lamb DC, Kim Y, Yermalitskaya LV, Yermalitsky VN, Lepesheva GI, Kelly SL, Waterman MR, Podust LM
(2006) Structure 14: 51-61
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Crystallography, X-Ray, Electron Transport, Flavin Mononucleotide, Flavin-Adenine Dinucleotide, Humans, Molecular Sequence Data, NADPH-Ferrihemoprotein Reductase, Rabbits, Rats, Saccharomyces cerevisiae Proteins, Sequence Alignment
Show Abstract · Added February 12, 2015
NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).
0 Communities
1 Members
0 Resources
14 MeSH Terms
Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase.
Guengerich FP
(2005) Arch Biochem Biophys 440: 204-11
MeSH Terms: Animals, Cytochromes b5, Cytochromes c, Detergents, Electron Transport, Kinetics, Lipids, Microsomes, Liver, NADPH-Ferrihemoprotein Reductase, Osmolar Concentration, Oxidation-Reduction, Phosphatidylcholines, Protein Binding, Rats, Transport Vesicles
Show Abstract · Added March 5, 2014
The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.
0 Communities
1 Members
0 Resources
15 MeSH Terms
Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes.
Shimada T, Mernaugh RL, Guengerich FP
(2005) Arch Biochem Biophys 435: 207-16
MeSH Terms: Coenzymes, Cytochrome P-450 Enzyme System, Cytochromes b5, Enzyme Activation, Enzymes, Immobilized, Ferredoxins, Humans, Kinetics, NADH, NADPH Oxidoreductases, NADPH-Ferrihemoprotein Reductase, Protein Binding, Protein Interaction Mapping, Pseudomonas putida, Substrate Specificity
Show Abstract · Added March 5, 2014
An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.
0 Communities
1 Members
0 Resources
14 MeSH Terms
Non-specific inhibition of human cytochrome P450-catalyzed reactions by hemin.
Kim EY, Kim JS, Kim MY, Koh WS, Guengerich FP, Yun CH
(2004) Toxicol Lett 153: 239-46
MeSH Terms: Catalysis, Cytochrome P-450 Enzyme Inhibitors, Enzyme Inhibitors, Hemin, Humans, Microsomes, Liver, NADPH-Ferrihemoprotein Reductase
Show Abstract · Added May 26, 2014
Hemin, a stable form of heme, is known to have an antimutagenic effect. Inhibitory effects of hemin on the cytochrome P450 (CYP)-catalyzed reactions of human liver microsomes and reconstituted systems containing purified CYP and NADPH-cytochrome P450 reductase (NPR) were seen. Hemin non-specifically inhibited all of the microsomal CYP activities examined. Hemin also inhibited 7-ethoxyresorufin O-deethylation, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin O-demethylation, and testosterone 6beta-hydroxylation catalyzed by purified CYPs 1A2, 2D6, and 3A4, with IC50 values of 27, 19, and 2.4 microM, respectively. Hemin also inhibited reduction of cytochrome c and ferricyanide by NPR, as much as 47%. Spectrally detectable CYP was destroyed in human liver microsomes and in a reconstituted system in the presence of hemin and an NADPH-generating system. We propose that the antimutagenic effect of hemin might be due to inhibition of CYP and NPR enzymes involved in the bioactivation of mutagens.
0 Communities
1 Members
0 Resources
7 MeSH Terms
Formation and reduction of aryl and heterocyclic nitroso compounds and significance in the flux of hydroxylamines.
Kim D, Kadlubar FF, Teitel CH, Guengerich FP
(2004) Chem Res Toxicol 17: 529-36
MeSH Terms: Carcinogens, Catalysis, Chromatography, High Pressure Liquid, Escherichia coli, Kinetics, Mass Spectrometry, Models, Theoretical, Mutagenicity Tests, NADP, NADPH-Ferrihemoprotein Reductase, Nitroso Compounds, Oxidation-Reduction, Quinolines, Salmonella typhimurium
Show Abstract · Added March 5, 2014
Cytochrome p450 (p450) 1A2 and NADPH-P450 reductase (NPR) catalyzed the oxidation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), with consumption of NADPH. The oxidation rate of NADPH by p450 1A2/NPR increased with time in the presence of IQ until depletion of NADPH. This unusual autocatalytic pattern of NADPH oxidation could be rationalized by formation of a nitroso derivative (IQ-N=O) and the subsequent reduction of the hydroxylamine (IQ-NHOH) and IQ-N=O, which would consume more NADPH. The formation of IQ-NHOH and IQ-N=O from IQ was confirmed using HPLC/MS. Reduction of IQ-NHOH and IQ-N=O was NPR-dependent but did not require p450. Autocatalytic NADPH oxidation was also observed in the oxidation of other heterocyclic and arylamines. However, the N-hydroxyl and nitroso oxidation products of 2-aminofluorene and 4-aminobiphenyl were reduced nonenzymatically by NADPH, and NPR did not catalyze the reactions. We simulated the enzymatic kinetic model for possible pathways for IQ metabolism, which included the formation of IQ-N=O, using some kinetic parameters obtained from the experimental results. In the kinetic model, we could reproduce the similar curvature for NADPH oxidation and the formation of IQ-N=O, and the reduction of IQ-NHOH and IQ-N=O is required to explain the observed results for NADPH oxidation. Our results support a role for nitroso derivatives of HAAs in the unusual autocatalytic NADPH oxidation and may have relevance in terms of possible toxicities of the nitroso derivatives. Both IQ-NHOH and IQ-N=O were mutagenic in a bacterial tester system devoid of p450 and NPR; the mutagenicity of both was decreased by expression of NPR, consistent with the reduction of these compounds observed with purified NPR.
0 Communities
1 Members
0 Resources
14 MeSH Terms
Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.
Henderson CJ, Otto DM, Carrie D, Magnuson MA, McLaren AW, Rosewell I, Wolf CR
(2003) J Biol Chem 278: 13480-6
MeSH Terms: Acetaminophen, Animals, Cytochrome P-450 Enzyme System, Exons, Female, Gene Deletion, Genomic Library, Glutathione, Liver, Male, Mice, Mice, Knockout, Mice, Transgenic, NADPH-Ferrihemoprotein Reductase, Pentobarbital, Promoter Regions, Genetic, Rats, Restriction Mapping, Serum Albumin, Sex Characteristics
Show Abstract · Added February 23, 2011
Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.
1 Communities
1 Members
0 Resources
20 MeSH Terms