Microsomal P450 2C3 Is Expressed as a Soluble Dimer inEscherichia coliFollowing Modifications of Its N-terminus

Wachenfeldt, Claes von; Richardson, Toby H.; Cosme, José; Johnson, Eric F.

doi:10.1006/abbi.1996.9859

Cited by 104 publications

(66 citation statements)

References 25 publications

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“…3). These results indicate that the N-terminal region of microsomal P450 is a critical region for membrane anchoring, as reported previously (57). dN2J3 was purified using the membrane fraction, since the specific content (P450/protein) was higher than that of the supernatant (Fig.…”

Section: Identification Of Female Rat Microsomal 25-hydroxylase By Pesupporting

confidence: 88%

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Yamasaki

Izumi

Ide

et al. 2004

Journal of Biological Chemistry

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Vitamin D 3 requires the 25-hydroxylation in the liver and the subsequent 1␣-hydroxylation in the kidney to exert its biological activity. Vitamin D 3 25-hydroxylation is hence an essential modification step for vitamin D 3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D 3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D 3 relative to other substrates. In this study, we purified vitamin D 3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D 3 and 1␣-hydroxyvitamin D 3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50 -60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D 3 and 1␣-hydroxyvitamin D 3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D 3 25-hydroxylation in rat liver. The most important physiological function described for 1,25-(OH) 2 D 3 is maintenance of calcium homeostasis (a process that involves calcium absorption from the intestine, reabsorption from the kidney, and mobilization from the bone) (2, 3, 5). In addition, involvement in a variety of processes such as cell differentiation and proliferation has been reported (1, 3). In the 1990s, two important kidney mitochondrial cytochrome P450s involved in vitamin D 3 metabolism were identified. One is CYP27B1, which catalyzes the 1␣-hydroxylation reaction, essential for vitamin D 3 activation. Its expression is regulated by parathyroid hormone, 1,25-(OH) 2 D 3 , and calcitonin (6 -11). The genetic defect of the gene results in pseudovitamin D deficiency rickets (12). The other is CYP24, which directs the catabolism of 1,25-(OH) 2 D 3 and 25-OH-D 3 by the hydroxylation at position C-24. The expression of this gene is primarily controlled by 1,25-(OH) 2 D 3 via a vitamin D receptor (13, 14). These two enzymes are apparently regulated in a reciprocal manner depending on physiological factors concerning calcium homeostasis, by which the biological activity of vitamin D 3 is maintained at appropriate levels in the body (1).In contrast to the 1␣-hydroxylase and 24-hydroxylase in kidney, the 25-hydroxylase of vitamin D 3 in liver remains not fully understood, although 25-OH-D 3 is the major circulation form of vitamin D ...

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Section: Identification Of Female Rat Microsomal 25-hydroxylase By Pesupporting

confidence: 88%

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Yamasaki

Izumi

Ide

et al. 2004

Journal of Biological Chemistry

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“…In contrast, based on gel filtration, isolated P450 2E1 was estimated to be in 10-mer complexes (34). Isolated P450 2B4 (34) and P450 2C3 (35) have also been shown to aggregate into complexes of hexamers and greater than octamers, respectively. Clearly, the aggregation of an isolated P450 in vitro does not correlate with the propensity to form homo-oligomers in the membrane of a living cell.…”

Section: Discussionmentioning

confidence: 95%

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Szczesna-Skorupa

Mallah

Kemper

2003

Journal of Biological Chemistry

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The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.The organization of the cytochrome P450 (P450) 1 -containing monooxygenase system in the microsomal membrane is not well understood despite many studies using a wide variety of biophysical methods. This system consists of P450s, NADPHcytochrome P450 reductase (P450 reductase), and for some P450s, cytochrome b 5 . If P450 levels in the endoplasmic reticulum (ER) are induced to maximal concentrations, there are 20 -30 P450s for each P450 reductase molecule. The presence of multiple forms of P450s in the ER membranes combined with limiting amounts of P450 reductase has important implications for the association of the P450s with each other and the reductase and for electron transfer from the reductase to P450. It has been postulated that either a multimeric complex of P450s binds to a molecule of P450 reductase (1, 2) or that the interaction results from random collisions of independently diffusing monomeric proteins that have high rotational and lateral mobility (3-5). Formation of large complexes by P450s has been observed with both isolated proteins and reconstituted systems (6 -8). Studies on rotational mobilities of P450s in microsomal membranes were also consistent with either self-aggregation or association of P450s with other components of the membranes (9 -11). The second model in which monomeric P450 interacts with P450 reductase is supported by observations that high concentrations of some P450s in membranes result in their aggregation and immobilization, but the addition of...

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“…[20][21][22][23] Based on this observation, a soluble and monomeric form of CYP2C5 and CYP2C3 was engineered by deleting the N-terminal membrane-spanning region. 24,25 Additionally, the N-terminus of CYP1A2 was shown to be involved in membrane anchoring and Nterminal modifications had an effect on the catalytic activity. 26 Interestingly, it was also demonstrated that membrane insertion of CYP1A2 was promoted by the presence of anionic phospholipids and this association with membrane is dependent on the Nterminus of CYP1A2.…”

Section: N-terminus Role In Association With Lipid Bilayers Activitymentioning

confidence: 99%

“…From literature, it is known that removal of hydrophobic regions from the N-terminus increases the yields of expression of cytochrome P450s. 25,46 Therefore, we prepared five different CYP2J2 constructs with varying N-terminal modifications for the purpose of increasing the yield of expression without eliminating their ability to associate with the lipid bilayers. The different N-terminally modified CYP2J2 constructs are listed in Figure 1(A), and the list of primers used for each respective construct are represented in Supporting Information Table S1.…”

Section: Construction Of Cyp2j2 N-deletion Mutantsmentioning

confidence: 99%

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

et al. 2013

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CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.

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Microsomal P450 2C3 Is Expressed as a Soluble Dimer inEscherichia coliFollowing Modifications of Its N-terminus

Cited by 104 publications

References 25 publications

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

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