A. General: Specific versus Multifunctional/Low-Specificity Cytochrome P450s

There are two major views of the functions of P450s. One is that these enzymes have specific and important physiological functions. The other view is that P450s have low specificity and, related to that, low catalytic activities; their function is to protect organisms (especially mammals, including humans) from natural products (Jakoby, 1980). Both views are correct, and P450s (at least mammalian P450s) fit into both categories (Table 1).

A number of heritable metabolic diseases are associated with loss of particular P450s, especially those involved in the metabolism of steroids and fat-soluble vitamins (Nebert and Russell, 2002). Studies with transgenic mice also support this view (Gonzalez and Yu, 2006). In addition, deletion of NADPH-P450 reductase is lethal to mouse embryos (Shen et al., 2002), although the exact reason is not clear [attenuation of the reductase in selected organs in mice has a relatively modest effect (Gu et al., 2003)]. Clinical issues have also been associated with polymorphisms in the reductase gene in humans (http://www.cypalleles.ki.se/por.htm). The P450s considered to be in this category generally do not vary considerably in levels of expression among individuals (Shinkyo and Guengerich, 2011) to the same extent as the “xenobiotic-metabolizing” P450s (Guengerich, 2005). Some of these P450s—but not all—have relatively high catalytic efficiencies (e.g., k_cat/k_m 2 × 10⁶ M⁻¹ s⁻¹ for purified cholesterol 7α-hydroxylase P450 7A1) (Shinkyo and Guengerich, 2011).

Mammals ingest considerable amounts of natural products (e.g., terpenes, alkaloids, flavonoids) in their food along with amino acids, carbohydrates, lipids, and so forth. Cells have two major means of protection: one is to transport the materials out (e.g., Mdr1 and related genes) and the other is metabolism. Thus, the presence of high levels of a set of enzymes (P450s) that are capable of removing a great variety of compounds from cells has a role in protecting cells from harmful chemicals. Drugs, pollutants, and so forth have structures that have some general resemblance to these natural products, at least in general terms of molecular weight, lipophilicity, polar surface area, and so forth. The information available to date indicates that even when these P450s (i.e., those using xenobiotics as substrates) do oxidize endogenous compounds (e.g., human P450 3A4 and testosterone hydroxylation), the reactions do not have important physiological consequences. For instance, many people (e.g., ∼ 7% of white persons) do not have P450 2D6 but seem to live normal lives. However, a caveat to this general view is that a small effect might have been missed in epidemiological studies, and a change of a few years of life expectancy or protection against some disease might have been missed.

Mammals have this less selective group of P450s to breakdown natural products, but the P450s in plants, which are much more numerous, seem to be devoted largely to anabolic reactions (i.e., generation of secondary metabolites that reduce infection, animal consumption, or enhance symbiotic interactions with other organisms for improved growth and reporduciton). For instance, several P450s have been identified in individual pathways to synthesize plant pigments and other secondary products (Humphreys and Chapple, 2000; Morant et al., 2003; Takei et al., 2004). Whether plants also have more general P450 systems for protection against xenobiotics is unknown; however, several cases of P450 involvement and adaptation to plant-insect “warfare” have been described previously (Gonzalez and Nebert, 1990; Schuler, 1996; Li et al., 2003). Common enteric bacteria do not have P450s (e.g., E. coli and Salmonella typhimurium). With more complex bacteria (e.g., Actinomyces spp.), the situation is less clear. Some functions of P450s have been described in the production of secondary products (Lamb et al., 2006), and there is also evidence that some can deal with a wide variety of xenobiotics (e.g., P450 105D1) (Taylor et al., 1999).

B. Classification by Substrates

Table 1 has already been discussed. However, the classification has some caveats. For instance, P450 1B1 oxidizes a wide variety of xenobiotics (Shimada et al., 1996) and also estrogens; this P450 is considered important in physiology in that polymorphisms have been shown to be associated with glaucoma (Stoilov et al., 1997; Vasiliou and Gonzalez, 2008). In addition, P450 27A1 oxidizes both cholesterol (a steroid) and vitamin D₃ and could be placed in either category. Many P450s that oxidize xenobiotics can also oxidize fatty acids (Niwa et al., 2009), but only a few P450s are included in this category in Table 1 when it seems to be a primary function for them. Exactly how critical these fatty acid reactions occur has not been established.

C. Major Cytochrome P450s Involved in Xenobiotic Metabolism

A. Instrumental Needs

The initial need is a system that can identify small differences between samples derived from two states (i.e., with and without active enzyme). The samples must be necessarily complex, either tissue extracts or body fluids. Therefore it is important to have a detection system that does not discriminate among molecules. Thus, UV and fluorescence are not really acceptable for initial screening.

¹H-NMR analysis has been used extensively in metabolomics studies (Bollard et al., 2005). However, resolution is limited. and most of the applications published so far involve changes in the patterns of major metabolites in body fluids, (e.g., amino acids, major carbohydrates, carboxylic acids of low relative molecular mass).

The most useful instrument for the purpose of identifying the functions of orphan enzymes is a mass spectrometer, for a number of reasons, including the following: the ability to resolve complex mixtures of compounds (either by LC coupling or high-resolution methods) (Fig. 2), 2) the generality of the detection mode (but see caveats below), and 3) the provision for annotation of candidate signals (e.g., using high resolution and/or fragmentation). There are several considerations about choices of mass spectrometry systems that need to be made, however, before commencing with analysis. One of the most important things to remember is that the goal in such work is to identify small changes between two complex mixtures that can be attributed to the action of a single enzyme, and all planning for instruments and informational analysis should be focused on this goal.

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Fig. 2.

LC-MS based strategies for elucidating substrates of human enzymes (Guengerich et al., 2010, 2011). One approach (in vivo) involves either expressing the human protein or deleting its apparent ortholog in a transgenic animal model. LC-MS metabolomic analysis can be done with body fluids from the animals or by removing tissues and using the in vitro incubations. Alternatively, a total in vitro approach can be used in which the protein is heterologously expressed and used with extracts of human tissue (with or without isotopically labeled cofactor). Data from the LC-MS analysis are used in comparisons (e.g., wild-type versus transgenic mice, in vitro enzyme incubations versus controls) made with one or more programs (e.g., PCA, DoGEX, MZmine, XCMS). The goal is to identify differences. In some cases, the mass spectrum (i.e., molecular formula) may be sufficient to identify possible matches of candidates, and the authentic materials may be available for comparison. However, if unknown compounds are involved, then not only the high-resolution MS spectra but also UV visible and NMR spectra may be needed. Ultimately, more experiments will be needed to assess the biological relevance of any findings.

In principle, it is possible to separate a complex mixture of compounds by direct injection into a high-resolution mass spectrometer (i.e., quadrupole time-of-flight, Orbitrap, or Fourier transform-MS). A spectrum would reveal the elemental formulae of all compounds (or at least provide a list of the best choices for each peak). Such approaches have been reported for annotation of the peaks in complex mixtures. However, there are at least three deficiencies associated with such an approach. One is the existence of isomers (i.e., for these purposes, compounds with the same elemental formulae). A second is that the lack of separation (in the LC phase) would lead to undue ion suppression in the MS phase. The third (linked with the second) is that comparing the quantities of individual compounds in the two samples is difficult.

More commonly, separation is done by GC and LC before introduction into the mass spectrometer. Capillary GC has excellent resolving power, but relatively few biological compounds (at least mammalian) are amenable to direct separation by GC; thus, derivatization is needed and imposes a preselection on which compounds will be analyzed.

A more common approach involves LC. Modern ultra-high-performance liquid chromatography systems have high resolving power, and typical reversed-phase methods have the capability to resolve low-molecular-weight biological molecules, particularly relatively nonpolar ones of the type that are P450 substrates. Typical water-to-acetonitrile gradients, with ammonium acetate (or formate), can be used with octadecylsilane (C₁₈)-based columns.

If compounds are not derivatized, then a choice must be made as to what ionization mode should be used for LC-MS work—positive ion electrospray ionization (ESI), negative ion ESI, or atmospheric pressure chemical ionization. Molecules bearing a positive charge (e.g., amines) ionize best with positive-ion ESI; molecules bearing a negative charge (i.e., carboxylic acids) ionize best with negative-ion ESI. Neutral molecules (e.g., steroids) are problematic and in our own experience work best with APCI. When one does not know what to expect in an LC-MS analysis, the most reliable approach is to do the analysis in all three modes. Obviously this increases the time and cost of analysis 3-fold.

Two approaches have been developed in our own laboratory to facilitate the analysis of P450 reaction products. Many of the products of P450 reactions are alcohols, although not all are (Guengerich, 2001; Isin and Guengerich, 2007; Guengerich and Isin, 2011). In the past, dansyl chloride has been used to convert amines and nucleophilic phenols to fluorescent derivatives. However, dansylated compounds also have excellent ionization properties in positive-ion ESI MS because of the positive charge on the amine moiety. We modified conditions (heat, use of 4,4-dimethylaminopyridine as a reagent) for derivatization of unactivated alcohols (Tang and Guengerich, 2010). The method was shown to be applicable to several alcohols that can be formed by the action of P450s on steroids, fatty acids, and vitamin D₃. In some cases, the limit of detection was lowered by >10³-fold (Tang and Guengerich, 2010). The products can be further defined by fragmentation, which removes the dansyl group.

Another approach we have used involves mixtures of ¹⁶O₂ and ¹⁸O₂ cofactors (Sanchez-Ponce and Guengerich, 2007). In this approach, the atmosphere of a P450 reaction is replaced with a 1:1 mixture of ¹⁶O₂/¹⁸O₂, resulting in oxygenated products (e.g., alcohols, epoxides) with a 1:1 mixture of ¹⁶O and ¹⁸O atoms, which should be detected as M/M + 2 doublets, as in the case of natural abundance ⁷⁹Br/⁸¹Br doublets. A MATLAB-based program (DoGEX) was developed to search for such doublets [alternatively, the program can be set to seek other isotopic products with other ratios (e.g., chlorine)] (Fig. 3) (Sanchez-Ponce and Guengerich, 2007). The program DoGEX also has features to allow baseline adjustment, noise removal, and calibration of retention times. With regard to technical aspects of handling ¹⁸O₂ gas, the procedures are relatively straightforward in that pressurized vessels of ¹⁸O₂ are now commercially available (e.g., Cambridge Isotope Laboratories, Andover, MA). Samples (of enzyme and a tissue extract) are placed in Thunberg tubes, and air is removed by alternate cycles of (mild) vacuum and exposure to argon gas. Isotopically labeled oxygen can be purchased as a 1:1 mixture of ¹⁶O₂ and ¹⁸O₂ (Cambridge Isotope Laboratories), although we have experienced unexplained problems with ¹⁶O-¹⁸O being present in some lots. A simpler system involves preparing separate/parallel samples with ¹⁶O₂ and ¹⁸O₂ and then mixing the contents after completion of the incubations.

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Fig. 3.

Use of the program DoGEX in screening for candidate substrates. Raw LC-MS data from an incubation of a P450, NADPH-P450 reductase, NADPH, a tissue extract, and a 1:1 mixture of ⁶O₂/¹⁸O₂ is shown in A. B shows the analysis of mass-to-charge ratio versus retention time, with a green positive spot on a yellow background for a ratio of ∼1.0 and Δ atomic mass units = 2.0, with the positive zone expanded in part C. The data from C are produced as a plot of relative ion intensity versus mass-to-charge ratio in part D. In this case the P450 uses P450 7A1, and the product was (the succinate ester of) 7α-hydroxycholesterol (Guengerich et al., 2010, 2011; Tang et al., 2010).

The approach has been validated using P450 3A4 and the substrate testosterone (Sanchez-Ponce and Guengerich, 2007). A more rigorous validation involved finding labeled 7α-hydroxycholesterol in an incubation of P450 7A1 and human liver extract (Fig. 3) (Tang et al., 2010). Fatty acid substrates were identified using the approach (with negative-ion ESI) with P450s 1A2, 2C8, and 2C9 (Tang et al., 2009) and 4F11 (Tang et al., 2010). Furthermore, the approach can be coupled with the dansylation procedure mentioned above; e.g., dansylated 7α-cholesterol could be identified in human liver extracts after incubation with P450 7A1 and ¹⁸O₂ (Tang and Guengerich, 2010). In principle, capillary electrophoresis-MS could be used as an approach (Dunayevskiy et al., 1996) but would probably yield a more restricted set of resolved analytes into the mass spectrometer (compared with LC).

B. Informatics

The goal of data processing is to identify a few major changes among thousands of compounds that distinguish the two different states. In the MS modes used in this work (see section III.A), the full scan mode is used across a wide mass-to-charge ratio range, resulting in very large data sets. Therefore it is unrealistic to analyze the data by visual inspection and specialized software is needed for this kind of data analysis.

Principal component analysis (PCA) is a well established general approach for comparing two or more data sets. PCA methods have been used widely in NMR metabolomics comparisons (Bollard et al., 2005) but apparently not extensively for MS approaches to enzyme function.

A number of freely available software programs have been developed to identify the differences between metabolite profiles acquired by on-line chromatography mass spectrometry techniques: XCMS (Smith et al., 2006), MZmine (Katajamaa et al., 2006), DoGEX (Sanchez-Ponce and Guengerich, 2007), MetAlign (Tolstikov et al., 2003), and MSFACTs (Duran et al., 2003). All of these programs are capable of adjusting small but problematic differences in retention time, one of the chief problems when comparing two (or more) complex LC-MS/full-scan profiles. In our laboratory, XCMS and MZmine have been the most widely used programs for untargeted metabolic profiling. We successfully identified an unusual endogenous dipentaenone substrate of P450 154A1 using XCMS and MZmine by comparing the metabolic profiles of wild-type and P450 154A1-knockout Streptomyces coelicolor (Fig. 4) (Cheng et al., 2010). DoGEX has already been mentioned. This program has been used in analyses involving isotope tags, and several fatty acids were verified as substrates of four hepatic P450s (1A2, 2C8, 2C9, 4F11) by this approach (Fig. 3) (Tang et al., 2009, 2010). In addition, some commercially available software is applicable to studies of this type [e.g., ACD/IntelliXtract (ACD/Labs, Toronto, ON, Canada), MetaboLynx (Waters, Milford, MA), and Mass Frontier (HighChem, Bratislava, Slovakia)].

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Fig. 4.

Use of an MZmine approach to identification of a substrate and product for S. coelicolor P450 154A1. LC-MS data were obtained for wild-type and P450 154AA1-knockout bacteria. Comparison of the LC-MS data by LC-MS using MZmine suggested a compound with MH⁺ 259. Extracts of the P450 154A1 bacteria (in which a substrate would be expected to accumulate) with P450 154A1 indicated that the candidate compound disappeared during incubation and a new peak appeared. Subsequent analytical work showed 1) the structures of the substrate QCP1 and product QCP2a,b (2 isomers) and 2) that no redox function was related to the change (Fig. 5) (Cheng et al., 2010).

C. In Vivo Approaches

There are inherently two approaches to experimental studies defining enzyme function, in vivo and in vitro. In vivo approaches will be discussed first, although this laboratory has only applied in vivo methods in the case of P450s in microorganisms. The discussion here will focus on mammals.

D. In Vitro Approaches

In vitro approaches have advantages in that the level of complexity can be reduced and, in principle, the mechanism underlying a phenotypic change may be seen directly.

E. In Vivo/In Vivo Combinations

Preliminary assays can be done in vitro or in vivo. However, combination approaches can also be used to advantage. In addition, it is important to emphasize that ultimately both in vitro and in vivo approaches are needed to understand mechanisms and to put the results into a physiological perspective.

1. Transgenic Animals and Tissue Extracts as Enzyme Sources.

One example comes from the work of Yu et al. (2003, 2004). Comparisons were made with liver microsomes derived from control mice and transgenic mice expressing human P450 2D6. Comparisons were done with a set of selected phenethylamines and indolethylamines. A number of these were shown not to be substrates, but two indolethylamines were—5-methoxy-N,N-dimethyltryptamine and picoline (6-methoxy-1,2,3,4-tetrahydro-β-carboline) (Yu et al., 2003, 2004). Furthermore, the catalytic efficiencies (k_cat/k_m) for P450 2D6 were 9 × 10⁴ and 2.4 × 10⁵ M⁻¹ s⁻¹, which can be considered respectable. However, in this approach, the human P450 2D6 is overlaid onto an endogenous background (of mouse 2d enzymes), so the somewhat orthologous mouse P450s must catalyze these reactions only at very low rates. The exact significance of these reactions in humans is unknown.

Another example comes from our work with Streptomyces spp. P450s (Cheng et al., 2010). A P450 154A knockout strain of S. coelicolor was compared with wild-type: extracts of each were compared by using LC-MS and the program MZmine (Fig. 4) (Katajamaa and Oresic, 2005; Katajamaa et al., 2006). This approach yielded a candidate, QCP1 (i.e., a putative substrate that accumulated in the knockout strain). An extract of this strain was also incubated with purified P450 154A1, which led to the disappearance of the peak of the substrate (QCP1) and the appearance of two new peaks as products (QCP2a,b) (Figs. 4 and 5) (Cheng et al., 2010). Thus, the in vitro approach was used to confirm the initial (in vivo) result. In this case, the knockout strain also had a discernable phenotype, the instability of spores in long-term storage. However, experiments have not been done to add the isolated substrate (or product) to the knockout strain in attempts to recover the wild-type phenotype. Nevertheless, this approach to combining in vitro and in vivo analysis should be applicable in any case in which a knock-out experimental animal model and a heterologously expressed protein are both available.

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Fig. 5.

Conversion of an endogeneous compund found in S. coelicolor to products without net oxidation-reduction (Cheng et al., 2010).

A. Cytochrome P450 2A7

mRNA transcripts have been reported in human liver samples (Ding et al., 1995; Koskela et al., 1999) and possibly esophagus (Godoy et al., 2002). Alternative splicing is apparently involved in producing P450 2A6/2A7 hybrid alleles (Koskela et al., 1999; Oscarson et al., 2002). The P450 2A6/2A7 “hybrid” allele has lower lower enzymatic activity than P450 2A6 (Oscarson et al., 2002). To date, attempts at heterologous expression of P450 2A7 protein in several systems have not produced active enzyme (Yamano et al., 1990; Ding et al., 1995), and it is not clear that an active form of P450 2A7 exists.

B. Cytochrome P450 2S1

mRNA expressed has been detected in human skin and liver, and there are also reports of expression in trachea, lung, stomach, small intestine, and spleen (Table 2). The gene is reported to be regulated by the aryl hydrocarbon receptor (Rivera et al., 2002, 2007).

Heterologous expression has been achieved in both yeast and E. coli systems, with relatively high levels in the latter case. The catalytic specificity of P450 2S1 has been controversial. Smith et al. (2003) reported that retinoic acid is a substrate for P450 2S1 but did not quantify a rate. Subsequently, the oxidation of retinoic acid has not been repeated in other attempts (Wu et al., 2006b; Nishida et al., 2010). Although P450 2S1 has been reported to be inducible via the aryl hydrocarbon receptor system, no carcinogens have been found to be substrates (Wu et al., 2006b). Bui and Hankinson (2009) reported that P450 2S1 could not be reduced by NADPH-P450 reductase and that several catalytic activities of P450 2S1 could be observed if reactions were supported by oxygen surrogates, e.g., hydroperoxides (Bui and Hankinson, 2009; Bui et al., 2009, 2010). Nishida et al. (2010) reported that P450 2S1 is capable of catalyzing the reduction of the substrate AQ4N, the di-N-oxide derivative of AQ4 [1,4-bis([2-(dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione], a topoisomerase-inhibiting drug. The rate was 12 min⁻¹, and a ferrous-carbon monoxide complex could be produced with the reductase (rate not measured). Therefore P450 2S1 seems capable of being reduced via electron flow from the reductase. A serious caveat in the work of Bui and Hankinson (2009) is that the reduction work was done aerobically, under conditions known to allow ferrous P450s to reoxidize rapidly (Guengerich et al., 1976). As a result of the delivery of oxygen from the capillaries, parts of organs are anoxic (e.g., reduction of CCl₄ by P450), and some tumors are rather anaerobic. Nishida et al. (2010) also point out that the conclusion of Bui et al. (Bui and Hankinson, 2009; Bui et al., 2009, 2010) that P450 2S1 normally uses lipid peroxides for its catalytic function is inconclusive, in that many P450s can react with lipid hydroperoxides through a shunt reaction that generates lipid alkoxy and peroxy radicals. These radicals can enter cooxidation reactions outside of P450 active sites (Mansuy et al., 1982; Ortiz de Montellano, 1995).

In conclusion, the only demonstrated reaction of P450 2S1 is reduction of AQ4N, and no oxidation reactions have been clearly established at this time. The rate of reduction of P450 2S1 by the reductase has recently been measured at 40 s⁻¹, and the AQ4N enhancement of NADPH oxidation has been demonstrated, clearly indicating the reduction of P450 2S1 (Xiao et al., 2011).

C. Cytochrome P450 2U1

mRNA has been detected in brain and thymus (Table 2) (Chuang et al., 2004; Karlgren et al., 2004). Heterologous expression of P450 2U1 in insect cells has been reported. The only reported reactions are hydroxylations of fatty acids, both ω- and ω-1 (Chuang et al., 2004).

D. Cytochrome P450 2W1

Attempts to find mRNA expression of P450 2W1 in human tissues were unsuccessful (Karlgren et al., 2005; Wu et al., 2006b), and expression seems to occur only in tumorous cells, particularly colorectal cancer (Gomez et al., 2010). Gomez et al. (2010) have also reported that a fraction of P450 2W1 expressed in human embryonic kidney 293 cells is glycosylated and also on the lumenal site of the endoplasmic reticulum, suggesting that it might not interact well with NADPH-P450 reductase.

P450 2W1 has been demonstrated to catalyze benzphetamine N-demethylation at a rate of ∼3 min⁻¹ (Wu et al., 2006b) and AQ4N reduction at a rate of 12 min⁻¹ (Nishida et al., 2010), when coupled with NADPH-P450 reductase. Very low rates of fatty acid oxidation have also been reported (Karlgren et al., 2006; Wu et al., 2006b). Some activity toward indole and indole derivatives has been observed (Wu et al., 2006b; Yoshioka et al., 2006; Gomez et al., 2010). Of particular significance is the ability of P450 2W1 to activate a variety of chemical carcinogens to genotoxic forms (Wu et al., 2006b), which may be of particular relevance in light of the localization of this P450 in cancers.

E. Cytochrome P450 3A43

This P450 is one of the four P450s in the 3A subfamily. Some controversy exists as to its significance, particularly as regards its importance relative to the other Subfamily 3A P450s, particularly the more dominant P450 3A4 (Guengerich, 1999). Nevertheless, 3A7 is generally expressed only in fetal tissue, many people do not express P450 3A5 because of polymorphisms (html://www.cypalleles.ki.se/), and some people do not appear to express active P450 3A4 (Guengerich and Turvy, 1991; Yang et al., 2010). mRNA levels of P450 3A43 were ∼0.1% of the mean levels of P450 3A4 and 2% the mean levels of P450 3A5 (Westlind et al., 2001). The localization of some of the mRNA in brain (Table 2) may be important, as well as a reported selectivity for oxidation of the drug alprazolam (Agarwal et al., 2008). Heterologous expression of active protein has been reported in E. coli (Domanski et al., 2001) and COS-1 cells (Agarwal et al., 2008) but not in yeast or mouse hepatic H2.35 cells (Westlind et al., 2001). Another substrate (with very low activity) is testosterone (Domanski et al., 2001).

F. Cytochrome P450 4A22

This gene does not seem to be expressed very extensively, with mRNA levels much lower than for P450 4A11 (Savas et al., 2003). This gene was originally named 4A11, but what was 4A22 (95% sequence identity) has now been renamed 4A11 and shown to be a dominant fatty acid ω-hydroxylase in human liver (Guengerich, 2005).

G. Cytochrome P450 4F11

The mRNA is expressed in liver (Table 2). Heterologous expression has been achieved in yeast, insect cells, and E. coli (Table 2). P450 4F11 has catalytic activity toward fatty acids and also β-keto fatty acids (Kalsotra et al., 2004; Dhar et al., 2008; Tang et al., 2010). No bioactivation was observed when a number of procarcinogens were tested (Tang et al., 2010). In recent studies, some activity in oxidation of vitamin K1 has been detected (A. E. Rettie, Y. Xiao, and F. P. Guengerich, unpublished results).

H. Cytochrome P450 4F22

Haplotype and gene mapping for nonsyndromic autosomal recessive congenital ichthyosis revealed a new gene (FLJ39501) on chromosome 19p12-q12. Seven mutations were identified in the gene, later identified as CYP4F22 (http://drnelson.uthsc.edu/CytochromeP450.html). mRNA expression was detected in skin and testis, with minor levels found in bone marrow, liver, small intestine, skeletal muscle, and brain Lefèvre et al. (2006) proposed that P450 4F22 is involved in the bioactivation of products derived from 12R-hydroperoxyeicosatetraenoic acid and 8R,11R,12R-epoxy-hydroxyeicosatrienoic acid. Nilsson et al. (2010) expressed P450 4F22 in Saccharomyces cerevisiae and reported that it oxidizes arachidonic acid to a mixture of hydroxyeicosatetraenoic acids.

I. Cytochrome P450 4V2

Mutations in the CYP4V2 gene (chromosome 4q35.1) have been found to be related to a rare eye disorder, Bietti crystalline corneoretinal dystrophy (BCD). This is an autosomal recessive chorioretinal dystrophy characterized by progressive night blindness (Li et al., 2004). Approximately 20 single-nucleotide polymorphisms have been reported, most of which have been discovered in Japanese or other Asian populations. The CYP4V2 gene consists of 11 exons, and mRNA has been found in human retina. Biochemical studies of patients with BCD indicate impaired fatty acid metabolism, which, along with homology to other P450 proteins, suggests that P450 4V2 may play a role in fatty acid and steroid metabolism (Li et al., 2004).

P450 4V2 has been expressed in insect cells and shown to catalyze ω-hydroxylation of medium-chain fatty acids (Nakano et al., 2009). However, the function is still unclear in that serum fatty acids and desaturase activites in BCD were not affected (in their subgroup analysis) by the different genotypes found in BCD patients (Lai et al., 2010), even though ocular crystal deposits were (Yokoi et al., 2010).

J. Cytochrome P450 4X1

An orthologous cDNA was cloned from rat brain (Bylund et al., 2002). Northern blotting indicated a specific expression to the brain, primarily in neurons of the hippocampus, cerebellum, and cortex. Full-length human P450 4X1 cDNA was cloned using human genome and expressed sequence tag databases, and the mRNA was found to be expressed in the trachea and aorta (Savas et al., 2005). The peroxisome proliferator activator receptor α has been reported to be involved in the activation of CYP4X1 gene transcription in a HepG2 cell line (Savas et al., 2005). In human brain, the distribution pattern shows a striking pattern with the highest levels in amygdala (Stark et al., 2008a). Human P450 4X1 could be expressed in E. coli and purified (Stark et al., 2008a). It is of interest that this P450 could catalyze a selective epoxidation of anandamide, a fatty acid amide, but not arachidonic acid (Stark et al., 2008a). However, the catalytic efficiency of the reaction was low, and the physiological significance of the product is unknown.

K. Cytochrome P450 4Z1

Transcriptome database screening for ESTs revealed the presence of P450 4Z1, a new P450, in a breast cancer cell line (SK-BR-3) (Rieger et al., 2004). P450 4Z1 mRNA expression is almost undetectable or at a low level in most human tissues, whereas abundant expression is noted in mammary tissue and breast carcinomas. It is still not known which cells express this P450 within breast tissue (Rieger et al., 2004; Savas et al., 2005). Expression has been confirmed in at least two known breast cancer cell lines, T47-D and MCF-7. In the progesterone-positive cell line T47-D, 15-fold induction of P450 4Z1 mRNA levels was found after treatment with progesterone, and a 10-fold induction after treatment with the glucocorticoid analog dexamethasone. This induction was suppressed by coadministration of the antagonist mifepristone (Savas et al., 2005).

The protein has been expressed in Schizosaccharomyces pombe (Zöllner et al., 2009) and in insect cells (Y. Xiao and F. P. Guengerich, unpublished observations). The only reported substrates are the fatty acids lauric acid and myristic acid (Zöllner et al., 2009), but the relevance of these reactions to any phenotype is unknown.

L. Cytochrome P450 20A1

mRNA expression has been detected in liver and brain, especially the hippocampus and substantia nigra (Stark et al., 2008b). It is noteworthy that a similar pattern is seen in rats. The significance of this localization is not known at this time. No substrates have yet been identified. It is of interest that related forms of this P450 appear in lower animals [e.g., as low as Xenopus tropicalis, fish, worms, sea anemone, and sponges (http://drnelson.uthsc.edu/cytochromeP450.html)].

M. Cytochrome P450 27C1

P450 27C1 mRNA is expressed in a number of tissues, including liver, kidney, and spleen (Wu et al., 2006a). The protein is in the P450 27 family, and the other two family members (P450s 27A1 and 27B1) are involved in vitamin D hydroxylation (Guengerich, 2005). P450 27A1 also hydroxylates cholesterol. P450 27A1 and 27B1 are mitochondrial enzymes and receive electrons from adrenodoxin reductase and adrenodoxin, and presumably P450 27C1 does as well. P450 27C1 did not oxidize vitamin D, 25-hydroxyvitamin D, or cholesterol (Wu et al., 2006a). No substrates have yet been identified. Recently a tricistronic approach has been developed for expressing P450 27A1 or 27C1, adrenodoxin reductase, and adrenodoxin together in E. coli (Salamanca-Pinzón and Guengerich, 2011) and may facilitate further searches for P450 27C1 reactions. It is noteworthy that mice do not have a P450 27C1 gene (but do require 27A1 and 27B1 for vitamin D homeostasis).