Expression of cytochrome P-450d by Saccharomyces cerevisiae

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae

Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.     

Identification of regions functioning in substrate interaction of rabbit liver cytochrome P-450 (laurate (omega-1)-hydroxylase)

The nucleotide sequence of cDNA for rabbit liver cytochrome P-450 (laurate (omega-1) hydroxylase) was replaced with that for rabbit liver cytochrome P-450 (testosterone 16 alpha-hydroxylase) in various regions coding for the amino acid sequence between residues 43 and 261. Six chimeric cDNAs thus constructed were cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. Chimeric P-450s synthesized in the transformed yeast cells were purified partially and their catalytic and spectral properties were examined and compared with those of the chimeric P-450 which is considered to possess the same catalytic properties as the wild-type P-450. In the oxidized state the chimeric P-450s exhibited a low-and high-spin mixed-type absorption spectrum of cytochrome P-450 and the spectrum was converted to a typical high-spin type on addition of laurate or caprate, indicating the binding of the fatty acids to the substrate site of the chimeric P-450s. However the affinities of the fatty acids for the chimeras devoid of the sequence of P-450 (laurate (omega-1)-hydroxylase) in either of the regions spanning residues 90-125 and 210-261 were 10 to 20 times lower than those for the chimeras containing the sequence of the wild-type P-450 in both regions. The latter chimeras have about the same affinities as the chimera which is essentially the wild-type P-450.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae

Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors. The relative efficiency of the phosphoglycerate kinase and GAL10-CYC1 promoters to direct the P-450 P1 mRNA synthesis was determined. The level of protein synthesis was found to be dependent on the amount of the 5'-noncoding sequence of the original cDNA removed during the construction. Yeast-synthesised P-450 P1 was found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities. The amount of P-450 reached up to 0.6% of the microsomal protein level. A nucleotide sequence coding for a chimeric enzyme in which 40 N-terminal codons of P-450 P1 were replaced by 36 N-terminal codons of P-450 P3 was constructed and expressed in yeast. The resulting protein retained full P-450 P1 activity and was produced with a similar efficiency suggesting that the P-450 N-terminal sequence is not involved in structures critical for the substrate specificities of the P1 isoenzyme.     

Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.     

Expression of rabbit cytochrome P-450IIE2 in yeast and stabilization of the enzyme by 4-methylpyrazole

A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.     

Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts

The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.     

Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity

The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B1 (AFB1). The most distinct effects were observed for gene conversion to tryptophan prototrophy. The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB1-induced mutagenicity indicates that activation of the promutagen AFB1 depends on the cytochrome P-450-catalyzed electron-transfer reactions. This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFB1-induced tryptophan conversion.     

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.     

Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene regulation in response to overexpression of cytochrome P450 and proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae

(CYP52A4) in Saccharomyces cerevisiae. Using the mRNA differential display technique, six genes were found to be up-regulated: ASN2, MDJ1, YLR194c, YNL208w, YER175, and YGL121c. Genes coding for Dur1.2p, Dal2p, and Sps19p were down-regulated. Two strongly induced genes, which were found to accommodate the peroxisome box (YLR194c) and a 10-bp consensus sequence of genes involved in lipid metabolism (YNL208w) in their promoter regions, were further analyzed with respect to the course of induction, the necessity of the P450 membrane anchor for induction, and the effects of gene disruption on P450Cm2 overexpression. We found that both genes are not essential to overproduce P450Cm2, but their induction was dependent on P450Cm2 membrane integration.     

Studies on the genetic regulation of cytochrome P-450 production in Saccharomyces cerevisiae

An initial survey of 18 haploid strains of Saccharomyces cerevisiae revealed that only 3 of these strains could produce a detectable level of cytochrome P-450. A cross between a cytochrome P-450 producing strain of S. cerevisiae (B/B) and a non-producing strain (D22) gave a diploid which was a non-producer and a 2:2 segregation of producers to non-producers in meiotic tetrads. Of the two producers in each tetrad, one produced a higher level of cytochrome P-450 than the other. We deduce that cytochrome P-450 production in S. cerevisiae is regulated by a single nuclear gene and that a modifier gene is also involved which can enhance the amount of cytochrome P-450 synthesized. Benzo(a)pyrene (an inducer of P-450 in yeast) had no effect on the action of the regulatory gene.     

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.     

Monooxygenase activity of Saccharomyces cerevisiae cells transformed with expression plasmids carrying rat cytochrome P-450MC cDNA

The recombinant plasmids pAMC1 and pJMC1 were constructed; the former contained the cytochrome P-450MC (P-450MC) cDNA expression unit consisting of yeast alcohol dehydrogenase I (ADH) promoter, rat P-450MC cDNA and ADH terminator, and the Leu 2 marker gene, and the latter contained the same expression unit and the leu 2-d gene. Saccharomyces cerevisiae AH22 cells transformed with each of the recombinant plasmids were examined for plasmid copy number, P-450MC mRNA level, P-450MC content, and monooxygenase activity. The S. cerevisiae AH22/pJMC1 cells contained about 2-fold higher levels of the plasmid, P-450MC mRNA, and P-450MC than the AH22/pAMC1 cells. Monooxygenase activity towards 7-ethoxycoumarin and acetanilide of the AH22/pJMC1 cells was 1.7-fold and 1.5-fold higher than that of the AH22/pAMC1 cells, respectively, whereas the activity of the AH22/pAMC1 cells towards 7-ethoxycoumarin and acetanilide was more than 1,000-fold 10-fold higher than that of the control AH22/pAAH5 cells which contain no P-450MC cDNA, respectively. Therefore, it is likely that monooxygenase activity of the AH22 cells carrying rat P-450MC cDNA was approximately proportional to the expression level of P-450MC cDNA.     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.     

cDNA cloning and expression of the mRNA for cytochrome P-450kd which shows a fatty acid omega-hydroxylating activity

We have recently purified three distinct forms of fatty acid omega-hydroxylase cytochrome P-450 (P-450), designated P-450ka-1, P-450ka-2 and P-450kd, from rabbit kidney cortex microsomes, and isolated and sequenced cDNA clones corresponding to P-450ka-1 and P-450ka-2 [Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Gotoh, M., Kusunose, M. & Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21,665-21,669]. The present paper describes cloning, sequencing and expression of a cDNA for the third fatty acid, omega-hydroxylase, P-450kd, from a rabbit kidney cDNA library. The cDNA for P-450kd encodes a polypeptide of 511 amino acids with sequence similarity of 87% to P-450ka-1. Its deduced NH2-terminal sequence of amino acids 5-24 is in complete agreement with the NH2-terminal sequence of P-450kd. The identity of the cDNA was further confirmed by its expression in COS-7 cells. When 14C-labeled lauric acid was added to the culture medium of COS-7 cells transfected with the cDNA, significant amounts of radioactive dodecanedioic acid, together with omega- and (omega-1)-hydroxylauric acids, were produced. Microsomes prepared from the transfected cells also efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid without formation of dodecanedioic acid. RNA blot analysis demonstrated that the mRNA for P-450kd gave a single band at the approximately 2.6-kb position. The mRNA for P-450kd was expressed in the liver and kidney, but not in many other tissues examined. Treatment of rabbits with clofibrate resulted in a elevated level of mRNA for P-450kd in both liver and kidney. Furthermore, the mRNA was remarkably increased in the kidney by the administration of cyclosporin A.     

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.     

Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus.

Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al. [D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6:1-11, 1987]). Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1. Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida. Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.