Roles of Divalent Metal Ions in Oxidations Catalyzed by Recombinant Cytochrome P450 3A4 and Replacement of NADPH-Cytochrome P450 Reductase with Other Flavoproteins, Ferredoxin, and Oxygen Surrogates

Yamazaki, Hiroshi; Ueng, Yune-Fang; Shimada, Tsutomu; Guengerich, F. Peter

doi:10.1021/bi00026a020

Cited by 131 publications

(114 citation statements)

References 33 publications

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“…In contrast, maximal EROD activity of the CYP1A2 system increased to a maximum at 200 mM HEPES and declined with further increases in buffer concentration. Declining activities with increasing buffer concentrations were expected and have been attributed to the disruption of electrostatic interactions between reductase and P450, consistent with previous studies (6)(7)(8)(9)(10)(11)(12)(13)(14)16,17,24,39). When comparing EROD in the mixed reconstituted system to the sum of the rates of the binary systems (which can be seen by comparing the third and fourth bars from each group), a significant synergistic stimulation of EROD was observed at lower ionic strength; higher concentrations of HEPES relieved this synergism.…”

Section: Resultssupporting

confidence: 91%

“…The observation of interactions in mixed reconstituted systems containing CYP1A2 (i.e. CYP1A2/CYP2B4 and CYP1A2/CYP2E1) taken together with the lack of detectable interactions with the CYP2B4/CYP2E1 system strongly suggests that some feature of CYP1A2 may facilitate the formation of functional interactions with other P450 enzymes.CYP1A proteins have previously been shown to interact with CYP3A subfamily P450s (13,21,24,40). Alston et al (40) provided evidence for a physical interaction between CYP3A2 and CYP1A1 by treating liver microsomes with a bifunctional crosslinking agent to covalently link closely associated proteins.…”

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confidence: 99%

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Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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Mixed reconstituted systems containing CYP2B4, CYP1A2 and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin-Odealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin-Odeethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.The cytochrome P450 (P450) superfamily of proteins is functionally diverse, and well distributed in nature. In addition to their roles in the metabolism of numerous endogenous compounds, P450s are important contributors to drug metabolism, primarily by catalyzing the insertion of an oxygen atom into a substrate molecule. NADPH cytochrome P450 reductase (reductase) is the major electron transfer partner, and is required for the majority of NADPHdependent oxidation reactions. Multiple forms of cytochrome P450 exist in the endoplasmic reticulum in a large excess over reductase (1), with the ratio of P450 to reductase dependent † These studies were supported by a US Public Health Service Research Grant from the National Institute of Enviromental Health Sciences ES004344 (WLB), and a predoctoral fellowship from the Stanley Scott Cancer Center (RWK). * Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, emailwbacke@lsuhsc.edu SUPPORTING INFORMATION AVAILABLE The supporting information includes ...

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Section: Resultssupporting

confidence: 91%

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confidence: 99%

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

2006

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“…Analogously, flavodoxin has been found to be absolutely (Refs. 19 and 24 and this report) and in one case, partially (25) required for P450 reactions supported by FNR-Fld systems. These results would indicate a functional similarity between the FMN-binding domain of P450 reductase and flavodoxins for electron transfer to microsomal cytochromes P450 as predicted by Porter and Kasper (10).…”

Section: Resultssupporting

confidence: 56%

Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

Jenkins¹,

Genzor²,

Fillat³

et al. 1997

Journal of Biological Chemistry

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“…Some evidence also suggests that b 5 functions as a modifier for some cytochrome P-450-catalyzed reactions although its mechanism is not clear (11,12). For example, b 5 stimulates the 6␤-hydroxylation of testosterone and nifedipine oxidation by recombinant CYP3A4 (13). It also augments C17-C20 lyase activity of pig, guinea pig, and human P-450 17␣ leading to predominant formation of androgens (androstenedione and dehydroepiandrosterone) over 17␣-hydroxylated steroids (progesterone and pregnenolone) (14 -16).…”

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confidence: 99%

Identification of Outer Mitochondrial Membrane Cytochrome b5 as a Modulator for Androgen Synthesis in Leydig Cells

Ogishima

Kinoshita

Mitani

et al. 2003

Journal of Biological Chemistry

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Outer mitochondrial membrane cytochrome b 5 is an isoform of microsomal membrane cytochrome b 5 . In rat testes the outer mitochondrial membrane cytochrome b 5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b 5 is undetectable. Outer mitochondrial membrane cytochrome b 5 present in the testis was localized in Leydig cells with cytochrome P-450 17␣ , which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b 5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b 5 stimulated the cytochrome P-450 17␣ -catalyzed reactions, 17␣-hydroxylation and C17-C20 bond 1 in the endoplasmic reticulum (ER), and the other is outer mitochondrial membrane cytochrome b 5 (OMb) (1-3). They consist of the following three domains: (a) the amino-terminal hydrophilic, (b) medial hydrophobic, and (c) carboxyl-terminal hydrophilic domains. A protoheme is bound to the first domain, which is highly conserved between b 5 and OMb with 70% identity (4, 5). The hydrophobic domain, consisting of about 20 amino acid residues, is embedded in the lipid bilayer and functions for the insertion of proteins into the membranes as tail-anchored proteins (6). The carboxyl-terminal 10 amino acid residues of b 5 are exposed to the luminal side of the ER cisterna (7,8) and are required for the targeting of the cytochrome to the organelle (9). The visible spectroscopic properties of the reduced forms are characteristic of the b 5 -type hemoproteins. OMb and b 5 are spectrographically indistinguishable from each other due to the highly conserved heme-binding portion (1, 2). OMb and b 5 have a similar mobility on SDS-PAGE and are co-purified unless specific precaution was taken in the purification procedures. Antibodies directed against b 5 that had been purified without the removal step of OMb cross-react with OMb. In some case, cross-reactions are observed even if a highly purified form of OMb or b 5 was immunized in rabbits. These facts made the discrimination between OMb and b 5 difficult. The most convincing way of the discrimination is use of the specific antibodies. We have such antibodies in a large collection of anti-OMb and anti-b 5 antibodies.Because b 5 is a hemoprotein with sixth co-ordination, it is incapable of activating an oxygen molecule as cytochrome P-450 does. One of its physiological functions is an electron transfer to terminal oxidases such as stearyl-CoA desaturase (10). Some evidence also suggests that b 5 functions as a modifier for some cytochrome P-450-catalyzed reactions although its mechanism is not clear (11,12). For example, b 5 stimulates the 6␤-hydroxylation of testosterone and nifedipine oxidation by recombinant CYP3A4 (13). It also augments C17-C20 lyase activity of pig, guinea pig, and human P-450 17␣ leading to predominant formation of androgens (androstenedione and de...

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Roles of Divalent Metal Ions in Oxidations Catalyzed by Recombinant Cytochrome P450 3A4 and Replacement of NADPH-Cytochrome P450 Reductase with Other Flavoproteins, Ferredoxin, and Oxygen Surrogates

Cited by 131 publications

References 33 publications

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

Identification of Outer Mitochondrial Membrane Cytochrome b5 as a Modulator for Androgen Synthesis in Leydig Cells

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