Enzymatic properties of human CYP2W1 expressed in Escherichia coli

doi:10.1016/j.bbrc.2006.04.080

Biochemical and Biophysical Research Communications

Volume 345, Issue 1, 23 June 2006, Pages 169-174

https://doi.org/10.1016/j.bbrc.2006.04.080 Get rights and content

Abstract

The human genome project revealed a new member of the P450 family 2, CYP2W1, which has orthologous form in other vertebrate species, suggesting CYP2W1’s significant physiological function. Recently, it was reported that CYP2W1 can metabolize arachidonic acid. In this study, we isolated human CYP2W1 cDNA, and successfully expressed truncated CYP2W1 lacking N-terminal 20 amino acids in Escherichia coli cells. In the bicistronic expression system for human CYP2W1 and NADPH-P450 reductase, the formation of blue pigment, indigo, was observed in bacterial cultures. Based on this result, we revealed that CYP2W1 catalyzes the oxidation of indole. In addition, CYP2W1 showed monooxygenase activity towards 3-methylindole and chlorzoxazone. However, no activity was observed towards fatty acids including arachidonic acid. Further analysis using an E. coli expression system will reveal substrate specificity of CYP2W1 and why this P450 isoform is universally conserved in vertebrates.

Section snippets

Materials and methods

Materials. A cDNA panel from different human tissues (Multiple Tissue cDNA (MTC™) Panels) was purchased from Clontech (Palo Alto, CA). Primer DNAs were purchased from Proligo Japan KK (Kyoto, Japan) or SIGMA GENOSYS (Hokkaido, Japan). The DNA sequencing kit, BigDye^® Teminator v3.1 Cycle Sequencing Kit was purchased from Applied Biosystems (Warrington, England). Pyrobest^® DNA polymerase and restriction enzymes were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). Human lung cDNA library No.

cDNA cloning and sequencing of human CYP2W1

The cloned CYP2W1 cDNA was 1473 bp in length containing one silent mutation at nucleotide position 166 (c → t). Thus, the cloned cDNA encodes 490 amino acids corresponding to the predicted amino acid sequence of human CYP2W1.

Distribution of CYP2W1 mRNA in human tissues

Real-time PCR analysis was performed to determine relative abundance of CYP2W1 in various human tissues. The cDNAs used for amplification were normalized based on the amounts of multiple housekeeping genes. As shown in Fig. 1, relative abundance of CYP2W1 mRNA was observed in

Discussion

Recently, Nelson et al. [1] reported that human CYP2W1 gene has some orthologous genes, mouse CYP2W1, dog CYP2W1, and cattle CYP2W1. They also predicted that orthologous genes are specific for endogenous substrates, whereas non-orthologous genes are likely to act on foreign substrates. They also suggested that human CYP2W1 is evolutionary older or has mutated more rapidly than the rest of the CYP2 genes in the light of phylogenetic analyses, indicating that human CYP2W1 might have been

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Expression of CYP2S1 and CYP2W1 in breast cancer epithelial cells and modulation of their expression by synthetic methoxy stilbenes
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CYP2W1 catalyzes the oxidation of indole and certain lipids including lysolecithin and their stereoisomers. Furthermore, it shows monooxygenase activity toward 3-methylindole and chlorzoxazone [17,18]. Moreover, this isoform was found to be involved in the metabolism of several procarcinogens, such as PAHs [19].
“Orphan” cytochromes are a new group of P450 cytochromes without a fully recognized biological role. The expression of these CYPs in tumors is higher than that in normal tissues, which makes them attractive as chemopreventive and/or therapeutic targets. In this study, we compared the effect of synthetic methoxy stilbenes and resveratrol on the expression of two orphan cytochromes, CYP2S1 and CYP2W1, in breast cancer cells.
Breast cancer cells, lines MCF7 and MDA-MB-231, were treated for 72 h with tested compounds. The expression of CYP2S1 and CYP2W1 was evaluated at the transcript and protein levels by RT-PCR and Western blot, respectively.
The constitutive expression of both isoforms was confirmed at the mRNA and protein levels. CYP2S1 and CYP2W1 showed higher expression in MDA-MB-231 cells. In MCF7 cells treated with stilbenes, the expression of both CYPs was increased at the mRNA level, whereas at the protein level this effect was confirmed for CYP2S1 alone. In contrast, in estrogen receptor-negative MDA-MB-231 cells treated with stilbenes, the expression of both CYPs decreased, but mostly at the transcript level.
The results of the present study confirmed the constitutive expression of CYP2S1 and CYP2W1 in breast cancer cells, although their relatively low level of expression suggests that they may be less involved in the transformation of therapeutic agents in these types of tumors. Stilbenes, particularly 3MS and 4MS, can modulate the expression of “orphan” CYPs more efficiently than resveratrol.
Biochemical Function of the Respiratory Tract: Metabolism of Xenobiotics
2018, Comprehensive Toxicology: Third Edition
The responses of the respiratory tract to xenobiotic exposure are often related to the numerous xenobiotic metabolizing enzymes expressed there, particularly the cytochrome P450 monooxygenases, which are the focus of this article. Since the publication of the ***second edition of this volume, new information on various relevant topics continued to emerge. These topics include the expression and regulation of additional P450 isoforms, species differences in their activities toward additional respiratory toxicants, and the association of genetic polymorphisms in their genes in humans with the susceptibility of the lung to environmental toxicants, as well as the availability of genetically engineered mouse models, including “humanized” models, that can be utilized to identify the in vivo functions of these enzymes in the metabolism and toxicity of xenobiotics. The outcomes of these new studies endorse the conclusion that the selective expression and activities of the P450 enzymes in anatomical regions and specific cells impart specific susceptibilities to these cells. They also provide insights to the potential influence of systemic metabolism on risks of xenobiotic toxicity in the respiratory tract, as well as possible regional specificity of the metabolic mechanisms.
Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids
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Human cytochrome P450 (P450) 2W1 is still considered an “orphan” because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn-1 18:1 LPC as a substrate much more efficiently than the sn-2 isomer; we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9R,10S) over (9S,10R). The kinetics and position specificities of P450 2W1-catalyzed oxygenation of lysophospholipids (16:0 LPC and 18:1 LPC) and fatty acids (C16:0 and C18:1) were also determined. Epoxidation and hydroxylation of 18:1 LPC are considerably more efficient than for the C18:1 free fatty acid.
Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers
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Previous reports concerning CYP2W1 expression were probably performed mainly in whole lung, explaining the very low levels detected [7,8]. The physiological significance of CYP2W1 expression in BM remains unknown, as the substrate specificity of CYP2W1 is still controversial [25,122]. CYP2W1 expression level was low in SCC and undetectable in AC.
Susceptibility to lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is largely influenced by the metabolic capacity of lung tissues. This capacity is partly determined by the expression profile of the cytochromes P450 (CYPs), a superfamily of enzymes that have relevant catalytic properties toward exogenous and endogenous compounds. Using quantitative real-time RT-PCR, we conducted a comprehensive analysis of the expression profile of the 57 human CYP genes in non-tumoral (bronchial mucosa and pulmonary parenchyma) and tumoral lung tissues of 18 patients with non-small cell lung cancer. This study highlights (i) inter-individual variations in lung expression for some CYPs, (ii) different CYP expression patterns between bronchial mucosa and pulmonary parenchyma, that indicate distinctive susceptibility of these tissues toward the deleterious effects of inhaled chemical toxicants and carcinogens, (iii) high intertumoral variability, that could have major implications on lung tumor response to anti-cancer drugs.
Molecular modeling of human cytochrome P450 2W1 and its interactions with substrates
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The human cytochrome P450 2W1 (CYP2W1) was categorized into the so-called “orphan” CYPs because of its unknown enzymatic function. However, recent studies showed that the recombinant CYP2W1 exhibited broad catalytic activity towards several chemicals. Furthermore, this enzyme was selectively expressed in some forms of cancers, whereas a very low expression was found in human normal issues. These render CYP2W1 as a potential drug target for cancer therapy. At present, however, little information is available on the active site topology and the substrate binding modes of CYP2W1. In this study, the three-dimensional model of CYP2W1 was constructed using the homology modeling method. Two known substrates, benzphetamine and indole, were then docked into the active site, and refined by molecular dynamics simulations. The interaction energy between the substrates and the enzyme was calculated and analyzed by using the MM-GBSA method. The results indicated that the constructed CYP2W1 model can account for the regioselectivity of this enzyme towards the known substrates and van der Waals interactions were the driving force for the substrate binding. Several key residues were identified to be responsible for the binding of indole and benzphetamine with CYP2W1. These findings provide useful information for the detailed characterization of the biological roles of CYP2W1 and structure-based drug design of this enzyme.
Targeting cytochrome P450 enzymes: A new approach in anti-cancer drug development
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Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e., inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of vitamin D₃ and vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.

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^☆: Abbreviations: CYP, cytochrome P450; Δ2W1, N-terminal truncated CYP2W1; 2W1, full-length CYP2W1; CPR, NADPH-P450 reductase.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

cDNA cloning and sequencing of human CYP2W1

Distribution of CYP2W1 mRNA in human tissues

Discussion

References (19)

De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase

J. Biol. Chem.

CYP2U1, a novel human thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids

J. Biol. Chem.

Cutaneous expression of cytochrome P450 CYP2S1: individuality in regulation by therapeutic agents for psoriasis and other skin diseases

Lancet

Expression patterns of mouse and human CYP orthologs (families 1–4) during development and in different adult tissues

Arch. Biochem. Biophys.

Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1

Biochem. Biophys. Res. Commun.

Novel extrahepatic cytochrome P450s

Toxicol. Appl. Phramacol.

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

J. Biol. Chem.

Purification of functional recombinant P450s from bacteria

Methods Enzymol.

High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli

Biochem. Pharmacol.

Cited by (32)

Expression of CYP2S1 and CYP2W1 in breast cancer epithelial cells and modulation of their expression by synthetic methoxy stilbenes

Biochemical Function of the Respiratory Tract: Metabolism of Xenobiotics

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers

Molecular modeling of human cytochrome P450 2W1 and its interactions with substrates

Targeting cytochrome P450 enzymes: A new approach in anti-cancer drug development

Enzymatic properties of human CYP2W1 expressed in Escherichia coli☆

Abstract

Section snippets

Materials and methods

cDNA cloning and sequencing of human CYP2W1

Distribution of CYP2W1 mRNA in human tissues

Discussion

J. Biol. Chem.

J. Biol. Chem.

Lancet

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Phramacol.

J. Biol. Chem.

Methods Enzymol.

Biochem. Pharmacol.