Secondary structure and membrane topology of cytochrome P450s

Tretiakov, V. E.; Degtyarenko, Kirill; Uvarov, V.Yu.; Archakov, Alexander I.

doi:10.1016/0003-9861(89)90389-5

Cited by 32 publications

(16 citation statements)

References 30 publications

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“…Measurements at high dilution (1 M) prevented us from observing the CD spectrum of the P450 1A2 in the near UV range. The secondary structures of salt-free and salt-induced P450 do not agree with values predicted from the method by Tretiakov et al (18).…”

Section: Resultsmentioning

confidence: 37%

Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Yun

Song

Ahn

et al. 1996

Journal of Biological Chemistry

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Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated sodium phosphate concentration. Here, the possible structural change of rabbit P450 1A2 accompanying the NaCl-induced increase in its enzyme activity is investigated by fluorescence spectroscopy, circular dichroism, and absorption spectroscopy. It was found that NaCl increased ␣-helix content and lowered ␤-sheet content of P450 1A2 in the presence as well as in the absence of a phospholipid. Intrinsic fluorescence emissions also increased with increasing NaCl concentration. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration in response to the increased salt concentration. The effect of increased potassium phosphate and NaCl on the P450 1A2 activity was also studied. It was found that the activity increase of rabbit P450 1A2 occurs concomitantly with the conformational change including raised ␣-helix content.Microsomal monooxygenase metabolizes a variety of endogenous and xenobiotic compounds. This enzyme system includes cytochrome P450 (P450) 1 enzymes and NADPH-P450 reductase, which transfers an electron from NADPH to P450. Cytochrome b 5 and NADH-cytochrome b 5 reductase may also contribute to the electron flow (2). Quite recently, Schenkman et al. (3) made an interesting observation that high salt concentration enhances the catalytic activity of rabbit P450 1A2. The P450 1A2 is one of various P450 enzymes that is induced by the polycyclic aromatic hydrocarbon. Since a high concentration of NaCl often stabilizes the proteins by preferential hydration (4), it is possible that this increased activity may accompany structural change of P450 1A2. It seems worthwhile, therefore, to study the molecular properties of structural changes of P450 1A2 in detail. Here, the hydroperoxide system instead of NADPH-P450 reductase and NADPH is used because it may be helpful in understanding the role of P450 1A2 conformation itself.We have studied the spectral change of the P450 brought about by salts and phospholipid in order to see whether the enhanced catalytic activity is related to the structural change of P450 1A2. The relationship between the structure and the activity is studied in detail for the first time in this study. The catalytic reactions by cumene hydroperoxide are studied in order to evaluate the role of the protein conformation in the catalytic function and to see how the environment of the heme is influenced by the conformational change induced by salts. The relationship between conformational changes and activities of the P450 is discussed. EXPERIMENTAL PROCEDURESChemicals-L-␣-Dilauroyl-sn-glycerol-3-phosphocholine (DLPC) and cumene hydroperoxide were from Sigma. 7-Ethoxycoumarin was obtained from Aldrich. 7-Ethoxyresorufin and resorufin were obtained from Molecular Probes, Inc. (Eugene, OR). Other chemicals were of the highest grade commercially available.Enzyme Sources-P450 1A2 was purified from liver microsomes of 5,6-benzoflavone-treated rabbits as described (5). The P450 ...

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Section: Resultsmentioning

confidence: 37%

Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Yun

Song

Ahn

et al. 1996

Journal of Biological Chemistry

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“…In the ER the Cyt P-450 and reductases are exposed to the cytosol. Mammalian Cyt P-450 appears to be anchored in the membrane by a 50 amino acid N-terminal sequence, with the heme-containing, catalytic domain on the cytosolic surface of the membrane (4,31). Cyt b5 and its reductase are held in the membrane by C-terminal anchors.…”

Section: Nadh:cyt B5 Reductase and Cyt B5mentioning

confidence: 99%

“…The mammalian sequences encode hemoproteins with (average) predicted structures consisting of46% a-helix, 12% ,8-sheet, 9% fl-turns, and 33% random coils. Two adjacent conserved f-a-fl units are recognized as a possible site of interaction with NADPH:Cyt P-450 reductase (31). Expression of the mammalian genes is under control at the transcriptional level, in response to xenobiotics, environmental conditions, and tissue-specific cues (22).…”

Section: Some Plant Cyt P-450 May Be Involved In Modification Of Herbmentioning

confidence: 99%

Multiple Forms of Plant Cytochromes P-450

Donaldson¹,

Luster²

1991

Plant Physiol.

159

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Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for 02 and NADPH. Several cytochromes P-450, Mr 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (Mr 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P-450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes, and perhaps mitochondria. A cytochrome P-450 demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P-450 from other organisms. The existence of cytochromes P-450 in different subcellular locations suggests that there are many genes for cytochromes P-450 in plants which have yet to be identified and classified.Cyt P-450 are membrane-bound heme-containing proteins which have been implicated in a variety of oxidative reactions in plant tissues. A wide range of reactions are mediated by specific forms of these proteins. Cyt P-450-linked enzymes have been implicated in biosynthetic pathways leading to the synthesis of lignin phenolics, membrane sterols, phytoalexins, and terpenoids (33). It has been postulated that plants have evolved highly specific Cyt P-450-linked secondary pathways to produce defense-related phytoalexins, while animals have evolved parallel less specific, Cyt P-450-linked systems to detoxify ingested phytoalexins and other xenobiotics (11,22 at the expense of the 450 nm peak (25). For this reason the CO-hemoprotein adduct with the 420 nm peak, termed P-420, is often considered to be a degradation product of Cyt P-450.In addition to the nonspecific inhibition by CO, certain Cyt P-450 activities are selectively inhibited by various imidazole, pyrimidine, and triazole derivatives. Inhibitor binding also can be determined spectrophotometrically by changes in difference spectra. A typical Cyt P-450 spectrum of any mem-669 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

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“…As a result, they often exhibit low (sometimes less than 20%) amino acid sequence identity (5,6). However, comparative analysis of known cytochrome P450 crystal structures (6, 7), hydropathy and secondary structure prediction algorithms (8), and molecular modeling (9) indicates that all P450s have a very similar spatial fold.…”

mentioning

confidence: 99%

Folding Requirements Are Different between Sterol 14α-Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs

Lepesheva

Podust

Bellamine

et al. 2001

Journal of Biological Chemistry

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Upon sequence alignment of CYP51 sterol 14␣-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting ␤-strand 3-2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways.

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Secondary structure and membrane topology of cytochrome P450s

Cited by 32 publications

References 30 publications

Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Conformational Change of Cytochrome P450 1A2 Induced by Sodium Chloride

Multiple Forms of Plant Cytochromes P-450

Folding Requirements Are Different between Sterol 14α-Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs

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