烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola

Three cytochrome P450 monooxygenases belonging to the CYP52 family were isolated from the genome of the sophorolipid-producing yeast Candida bombicola using degenerate PCR and genomic walking. One gene displayed high identity with the CYP52E members and was classified into this group ( CYP52E3 ), whereas the other genes belonged to new groups: CYP52M and CYP52N. CYP52E3 and CYP52N1 turned out to be of no relevance for sophorolipid production, but show clear upregulation when the yeast cells are grown on alkanes as the sole carbon source. On the other hand, CYP52M1 is clearly upregulated during sophorolipid synthesis and very likely takes part in sophorolipid formation.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Structure-activity relationships of organosulfur compounds as inhibitors of cytochrome P450 1-mediated activation of benzo[a]pyrene in a human cell model

Twelve naturally-occurring organosulfur compounds were investigated as inhibitors of cytochrome P450 1 (CYP450 1)-mediated activation of benzo[a]pyrene (B[a]P) in human hepatoma (HepG2) cells. Inhibition depended on the presence of a diallyl group and the number of S atoms. Diallyl trisulfide (DATS), with a diallyl group and three S atoms, had the highest activity with an IC₅₀ of 0.4 mM, and 1.5-fold higher potency than diallyl disulfide (DADS) containing a diallyl group and two S atoms. Organosulfur compounds containing an alkyl group were less effective, or even ineffective, inhibitors of both CYP450 1 and B[a]P-induced cytotoxicity than DADS and DATS. Alliin and S-allyl cysteine containing the S-cysteinyl group had no inhibition.     

Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (-)-alpha-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (-)-alpha-pinene to its regioisomer (-)-beta-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (-)-alpha-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

Catalytic and immunochemical properties of hepatic cytochrome P450 1A in three avian species treated with β-naphthoflavone or isosafrole

Induction of cytochrome P450 1A (CYP1A) can be used as a biomarker of exposure to planar halogenated aromatic hydrocarbons (PHAHs). Our objective was to characterize the induction of CYP1A activity and protein in three avian species following in vivo treatment with β-naphthoflavone (BNF) and/or isosafrole. Alkoxyresorufin-O-dealkylase (alk-ROD) activities of hepatic microsomes from Herring Gulls (Larus argentatus) (HGs), Double-crested Cormorants (Phalacrocorax auritus) (DCCs) and chickens (Gallus domesticus) were measured using ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin, in the presence and absence of the inhibitors ellipticine or furafylline. Immunoreactivity of microsomal proteins with antibodies to several CYP1A proteins was investigated. CYP1A protein and alk-ROD activities of HGs and DCCs, but not chickens, were induced by isosafrole. Ellipticine was a potent and non-selective inhibitor of alk-ROD activity in all three species, while furafylline inhibition of alk-ROD activities varied among species and treatments. In all three species, BNF induced a protein immunoreactive with monoclonal antibody to CYP1A1 from the marine fish Stenotomus chrysops (scup), but a CYP1A2-like protein was not detected in avian microsomes probed with polyclonal antibodies to mouse CYP1A2. Variations in responses among avian species indicate that CYP1A proteins and substrate specificities should be characterized for each species used in PHAH biomonitoring programs.     

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self‐limiting, due to P450 inactivation; therefore, the inhibition of CYP‐specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2‐catalyzed reactions (2β‐ and 6β‐testosterone hydroxylation) are very efficiently inhibited; CYP2C11‐ and CYP2B1/2‐catalyzed reactions (2α‐ and 16β‐testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1‐, CYP2A1/2‐, and CYP1A1/2‐related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40–50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital‐, and dexamethasone‐treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital‐treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital‐treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 53–61, 1999     

Inhibition of benzo[a]pyrene-induced cytotoxicity and cytochrome P450 1A activity by dietary flavonoids in human liver cell model: structure-activity relationship

Inhibition of benzo[a]pyrene (B[a]P)-induced cytotoxicity and cytochrome p450 1A (CYP 1A) activity by flavonoids (1–100 M) was examined in terms of the structure-activity relationship in the human liver-derived cell model (HepG2). Two hydroxyl groups in the 5- and 7-position of flavonoids were essential to inhibit B[a]P-induced cytotoxicity. Generally, flavones (IC₅₀; 5.0–17.2 M) were more potent than the corresponding flavonols (IC₅₀; 42.7–131.8 M), and flavonoids such as apigenin (IC₅₀; 7.2 M) were more active than the corresponding isoflavonoids, genistein (IC₅₀; 61.7 M). The planar structure of flavone proved to be important in inhibiting B[a]P-induced toxicity and CYP 1A activity. The inhibitory effect of flavonoids on B[a]P-induced CYP 1A activity was correlated well with the inhibition of B[a]P-induced cytotoxicity (r=0.635, p<;0.01).     

Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis

CYP1A1 homology models based on the CYP2C5 and a composite of CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the recently published coordinates of CYP1A2. The model using the CYP1A2 coordinates, CYP1A1-(1A2), gave near ideal stereochemical quality and was favored energetically. Docking studies identified the active-site residues potentially involved in binding of the prototypic CYP1A1 substrate 7-ethoxyresorufin. CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresrufin in a catalytically favorable orientation. Although 7-ethoxyresorufin O-deethylation by CYP1A1 and several mutants exhibited substrate inhibition, it is unlikely that inhibition arises from the simultaneous binding of two substrates within the active-site given the geometry of the active site-cavity.     

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-depen-dent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver :body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate ω- and ω-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-de-alkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Induction of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium by a barbiturate analog, 1-[2-phenylbutyryl]-3-methylurea

Summary In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843–850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.Abbreviations PBMU 1-[2-phenylbutyryl]-3-methylurea - M.P. melting point     

Molecular cloning and characterization of cytochrome P450 and ferredoxin genes involved in bisphenol A degradation in Sphingomonas bisphenolicum strain AO1

Aims: To clone and characterize the genes bisdA and bisdB, encoding Ferredoxin_bisd (Fd_bisd) and cytochrome P450_bisd (P450_bisd), respectively, from the bisphenol A (BPA) degrading Sphingomonas bisphenolicum strain AO1. Methods and Results: The 3·7 kb region containing bisdA and bisdB was cloned by genome walking and colony hybridization. The deduced N-terminal amino acid sequences of bisdA and bisdB were consistent with those of Fd_bisd and P450_bisd proteins characterized in our previous report. Two transposase genes, tnpA1 and tnpA2, were also located upstream and downstream of bisdAB. From amino acid sequence analysis, P450_bisd has two conserved regions corresponding to the oxygen and heme binding regions of the bacterial cytochrome P450 family. Fd_bisd was similar to putidaredoxin-type [2Fe-2S] ferredoxins. Escherichia coli BL21 (DE3) cells bearing bisdB- and bisdAB-recombinant pET19b were able to degrade BPA. A spontaneous mutant, strain AO1L, which was unable to degrade BPA, was isolated from the stock culture, and it was confirmed that strain AO1L had no bisdAB region. Conclusions: P450_bisd monooxygenase sytem, encoded by bisdAB, is one system required for BPA hydroxylation in S. bisphenolicum strain AO1. Significance and Impact of the Study: Our results indicate that bisdAB are key genes for BPA degradation in S. bisphenolicum strain AO1.     

Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis

Fusarium Tri4 encodes a cytochrome P450 monooxygenase (CYP) for hydroxylation at C-2 of the first committed intermediate trichodiene (TDN) in the biosynthesis of trichothecenes. To examine whether this CYP further participates in subsequent oxygenation steps leading to isotrichotriol (4), we engineered Saccharomyces cerevisiae for de novo production of the early intermediates by introducing cDNAs of Fusarium graminearum Tri5 (FgTri5 encoding TDN synthase) and Tri4 (FgTri4). From a culture of the engineered yeast grown on induction medium (final pH 2.7), we identified two intermediates, 2alpha-hydroxytrichodiene (1) and 12,13-epoxy-9,10-trichoene-2alpha-ol (2), and a small amount of non-Fusarium trichothecene 12,13-epoxytrichothec-9-ene (EPT). Other intermediates isotrichodiol (3) and 4 were identified in the transgenic yeasts grown on phosphate-buffered induction medium (final pH 5.5-6.0). When Trichothecium roseum Tri4 (TrTri4) was used in place of FgTri4, 4 was not detected in the culture. The three intermediates, 1, 2, and 3, were converted to 4,15-diacetylnivalenol (4,15-diANIV) when fed to a toxin-deficient mutant of F. graminearum with the FgTri4+ genetic background (viz., by introducing a FgTri5- mutation), but were not metabolized by an FgTri4- mutant. These results provide unambiguous evidence that FgTri4 encodes a multifunctional CYP for epoxidation at C-12,13, hydroxylation at C-11, and hydroxylation at C-3 in addition to hydroxylation at C-2.