New mathematical procedure for analysis of data obtained by means of circular dichroism titration method

A hypervalent iron-oxene species has been widely proposed as the "active oxygen" in cytochrome P450 (P450)-catalyzed reactions. We recently examined the effect of mutation of the highly conserved threonine residue in P450s 2B4 and 2E1 to alanine, a change that is believed to interfere with proton delivery to the active site, and have determined the change in rates of deformylation of aldehydes, epoxidation of olefins, and hydroxylation of various substrates. The results support the concept that three distinct oxidants are functional in P450 catalysis: nucleophilic peroxo-iron, nucleophilic or electrophilic hydroperoxo-iron, and electrophilic oxenoid-iron. The occurrence of multiple oxidizing species may contribute to the remarkable versatility of the P450 family of isozymes in the modification of drugs and other substrates. Furthermore, the relative concentrations of these oxidants in a particular P450 isozyme may contribute to substrate specificity and govern the type of reaction catalyzed.     

Stereochemistry of the biotransformation of 1-hexene and 2-methyl-1-hexene with rat liver microsomes and purified P450s of rats and humans

The epoxidation of 1-hexene (1a) and 2-methyl-1-hexene (1b), two hydrocarbons present in the ambient air as pollutants, is catalyzed by some human and rat P450 enzymes. The enantioselectivities of these processes, when the reactions were carried out using rat and human liver microsomal preparations, were modest and dependent on both P450 composition and substrate concentrations. Various P450 isoforms (rat P450 2B1 and human P450 2C10 and 2A6) catalyzed the double bond oxidation of 1a and 1b with different product enantioselectivities. In the case of 1a, a moderately enantioselective hydroxylation at the allylic C(3) with the formation of 1-hexen-3-ol (4a) by microsomes from control or preinduced rats was also observed. The oxidation of this metabolite was, in turn, catalyzed by rat liver microsomes and mainly by rat P450 2C11, leading exclusively to the formation of 1-hexen-3-one, with no double bond epoxidation being observed. The stereochemical course of the microsomal epoxide hydrolase-catalyzed hydrolysis of the epoxy alcohols, threo-(+/-)- and erythro-(+/-)-1, 2-epoxyhexan-3-ol, theoretically expected to be formed from 4a, has been investigated.     

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.     

Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s

Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7alpha-, 6beta- and 2alpha-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2'-isopropyloxy-4'-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.     

The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11 alpha-hydroxy groups (11 alpha-OH-testosterone and 11 alpha-OH-cortisol) decreased leakage, and those with 11 beta-OH groups (11 beta-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.     

Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the p450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.     

Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation

Mutations at amino acid positions 107-120, which are part of a predicted substrate recognition site [Gotoh, O. (1992) J. Biol. Chem. 267, 83-90], were analyzed in C2MstC1, a chimera of P450 2C2 and P450 2C1. This hybrid protein has a new activity for progesterone C21-hydroxylation in addition to the lauric acid (omega-1)hydroxylase activity present in both parent proteins. Various substitutions for highly conserved glycines at positions 111 and 117 and tryptophan at position 120 strongly decreased the lauric acid hydroxylase activity of P450 2C2 and C2MstC1 and the progesterone hydroxylase activity of C2MstC1. Activities of mutant proteins with substitutions at 107, 108, and 112-115 were also strongly reduced. Modest or no decreases in activity were observed for substitutions at 109, 110, 116, 118, and 119. Lauric acid hydroxylase activity decreased more in most C2MstC1 mutants than in those of P450 2C2, particularly at positions 107 and 108. A substitution of phenylalanine for valine-112 reduced progesterone hydroxylation by 30-fold while only moderately reducing lauric acid hydroxylase by 40%. This differential effect on two dissimilar substrates demonstrates the importance of residue 112 for substrate interactions. The results are consistent with a model in which residues 107-110 align with the B'-helix of the bacterial proteins P450cam and P450BM-3. This helix is followed by a substrate-contacting loop from 111 to 116, and residues 117-120 align with the C-helices of the bacterial proteins In this alignment, Trp-120 is positioned behind the heme such that it could participate in electron transfer from reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time interval effect on repeat cervical smear results

Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants. In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane. Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions. For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions. The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively. The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate. A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions. In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions. Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s. However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes. The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms.     

The catalytic site of cytochrome P4504A11 (CYP4A11) and its L131F mutant

CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.     

Mitochondrial glutathione: importance and transport

1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and 1H-nmr, and was consistent with a general metabolic pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C4', and not at the C3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P450 isozymes seem to be involved in flavonoid demethylation.     

Comparison of laparoscopic versus open repair of paraesophageal hernia

Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16alpha-position but was less active in estrone 16alpha-hydroxylation; the latter reaction and also estradiol 16alpha-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16alpha-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16alpha-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. alpha-Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, alpha-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis [Service, R. F. (1998) Science 279, 1631-1633].     

An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.     

Studies on the effect of intratesticular administration of opioid peptides, naloxone or N-acetyl beta-endorphin antiserum on some testicular parameters in rats

Substrate specificity and other properties of a fatty acid monooxygenase system in kidney microsomes of the Japanese house musk shrew (Suncus murinus) were examined. The suncus kidney microsomes catalyzed the hydroxylation of various saturated and unsaturated fatty acids to the omega- and (omega-1)-hydroxy derivatives. Laurate was most effectively hydroxylated among saturated and unsaturated fatty acids. The specific activity (53.79 +/- 5.59 [mean +/- SD, n = 6] nmol/nmol cytochrome P450/min) of laurate in suncus kidney microsomes was very high compared with that in liver and kidney microsomes of other species. C18 unsaturated fatty acids were converted to epoxides by a cytochrome P450-dependent fatty acid monooxygenase system in suncus kidney microsomes, in addition to omega- and (omega-1)-hydroxylation products. The monooxygenase system metabolized arachidonic acid only to omega- and (omega-1)-hydroxylation products, not to epoxidation products.     

Cryoglobulinemia and chronic liver diseases]

Taxotere, a promising anticancer agent, is metabolized almost exclusively in liver and excreted from bile in all species. To determine which cytochrome P450 is involved in taxotere biotransformation, 11 cDNA-expressed human cytochrome P450s were examined for their activity in the metabolism of taxotere and its derivatives. Of all P450s, cytochrome P450 3A4 and 3A5 were the most active for the oxidation of taxotere to the primary metabolite RPR104952 and for subsequent oxidation of RPR104952 to RPR111059 and RPR111026. RP70617, an epimer of taxotere was also metabolized by both P450 3A enzymes to form metabolite XII. The activity of 3A4/5 enzymes for these substrates was 4-50-fold greater than the other P450s examined. The Kms of 3A4 and 3A5 for taxotere were 0.91 and 9.28 microM, and Vmax for the formation of RPR104952 were 1.17 and 1.36 m(-1), respectively. The contribution of the 3A enzyme complex to the metabolism of taxotere in human livers from 21 individuals was assessed with the inhibitory monoclonal antibody and ranged from 64-93%. The primary oxidative metabolism of taxotere by human liver microsomes was well correlated with 3A4-dependent reactions for testosterone 6beta-hydroxylation (r2 = 0.84), taxol aromatic hydroxylation (r2 = 0.67) and aflatoxin B1 3alpha-hydroxylation (r2 = 0.63); whereas a poor correlation was found for reactions specifically catalysed by other P450s (all r2 < or =O.17). The extent of taxotere metabolism also closely correlated with levels of 3A4 enzyme in human livers quantified with immunoblot monoclonal antibody (r2 = 0.61). These results demonstrate that the P450 3A4 and 3A5 enzymes are major determinants in taxotere oxidation and suggest that care must be taken when administering this drug with other drugs that are also substrates for these enzymes.     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Down-regulation of the expression of three major rat liver cytochrome P450S by endotoxin in vivo occurs independently of nitric oxide production

Endotoxemia results in both the down-regulation of multiple cytochrome P450 genes and the induction of inducible nitric oxide synthase (NOS2). The nitric oxide (NO) released during inflammation has been implicated as the mediator of the decreased catalytic activity and expression of several cytochrome P450 isozymes. We examined the role of NO in the decreases of both gene expression and activity of three major P450s in the endotoxemic Fischer 344 rat. Endotoxin (LPS) treatment suppressed both mRNA and protein expression of P450 2C11, 2E1, and 3A2. Coadministration of the NOS inhibitor aminoguanidine to LPS-treated rats completely inhibited the release of NO into the plasma but did not reverse the down-regulation of expression of any of the P450s examined at three time points. LPS treatment had a biphasic effect on some P450 catalytic activities. The hydroxylation of testosterone at the 2alpha-, 16alpha- and to a lesser extent 6beta-positions, was inhibited 6 hr after LPS treatment and returned to normal by 12 hr. The role of NO in the 6 hr effects could not be assessed due to effects of the aminoguanidine treatment itself. The second phase of decreased P450 activities seen after 24 hr was attributed to the NO-independent decrease in gene expression. Our results suggest that NO is not required for the LPS-evoked down-regulation of P450 2C11, 2E1 and 3A2 mRNA or protein expression. We cannot rule out a possible role for NO in the decreases in P450 activities seen early in the response.     

Primary oxidation mechanisms in degradation of aliphatic hydrocarbons by bacterial enzyme systems (author's transl)

The bacterial dissimilation of aliphatic hydrocarbons is catalysed by a monooxygenase mechanism with incorporation of molecular oxygen. Numerous publications have shown the cytochrome P 450-dependent hydroxylation of hydrocarbons, but there is considerably less information of hemo-protein-independent hydroxylations by alkanhydroxylases. In a marine Pseudomonad we found a system sensitive to cyanide: The oxygenase could be divided into three protein fractions. A cytochrome P 450 type spectrum was not detected. The NADH-dependent hydroxylation of n-decane can be activated by Mg2+ and Fe2+ ions. A noncompetitive product inhibition occurs which deserves special attention. An alcohol-dehydrogenase is closely associated with the oxygenase system by a kind of multienzyme-complex. Studies on kinetics and substrate specificity of this enzyme show an inhibition by excess substrate increasing with the chain length of the alcohols. The whole complex (alkanhydroxylase, alcoholdehydrogenase and aldehyddehydrogenase) is induceable by bacterial growth on alkanes, primary alcohols and fatty acids as sole carbon source.     

Ethanol interactions with other cytochrome P450 substrates including drugs, xenobiotics, and carcinogens

Chronic ethanol abuse is associated with increased activity of the microsomal ethanol-oxidizing system. This effect is due primarily to induction by ethanol of a specific cytochrome P450 (CYP2E1) responsible for enhanced oxidation of ethanol and other P450 substrates and, consequently, for metabolic tolerance to these substances. Furthermore, cytochrome 450 induction increases the activation of numerous xenobiotics to toxic metabolites and of chemical carcinogens to reactive metabolites, thereby accelerating their adverse effects. Microsomal enzyme induction has been associated with increased reactive oxygen species production and enhanced lipid peroxidation, as well as with decreased enzymatic and nonenzymatic scavenger activity, providing another possible explanation for ethanol-mediated toxicity. Yet another effect of chronic alcohol abuse is chronic immune system activation, which is the mechanism underlying alcohol-related liver disease. The metabolism of steroids and vitamins is catalyzed by P450 and is altered in chronic alcoholics. This article reviews recent advances in the understanding of ethanol interactions with drugs, toxic agents, and carcinogens, as well as with steroids and vitamins.     

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.     

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

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.