Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium

The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995).     

Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for functional and structural multiplicity of pregnenolone-16 alpha-carbonitrile-inducible cytochrome P-450 isozymes in rat liver microsomes

Administration of pregnenolone-16 alpha-carbonitrile (PCN) to adult female rats caused a 2-fold increase in total liver microsomal cytochrome P-450 along with 5-7-fold increases in four in vitro monooxygenase activities considered diagnostic for the major PCN-inducible cytochrome P-450 isozyme. However, upon administration of chloramphenicol to PCN-treated rats, these monooxygenase activities could be resolved into three groups. Thus, the ability of the microsomes to convert triacetyloleandomycin to a metabolite that forms a spectral complex with the reduced heme iron was decreased by 80% by chloramphenicol, whereas only a 50% decrease was observed in the rate of conversion of (R)-warfarin to its 9,10-dehydro metabolite and in the rate of 6 beta-hydroxylation of androstenedione. More strikingly, the 10-hydroxylation of (R)-warfarin was actually enhanced 2-fold by the chloramphenicol treatment. Fractionation studies were carried out on liver microsomes from PCN-treated adult male rats, and two highly purified cytochromes P-450, referred to as PCNa and PCNb, were recovered. PCNb was found to be identical in the sequence of the first 15 amino acid residues with a PCN-inducible isozyme, the complete amino acid sequence of which has recently been deduced in another laboratory [Gonzalez, F. J., Nebert, D. W., Hardwick, J. P., & Kasper, C. B. (1985) J. Biol. Chem. 260, 7435-7441]. The other isozyme, PCNa, differed in amino acid sequence in three of the first 15 positions from PCNb. Upon immunoblot analysis, polyclonal antibodies raised to PCNb also recognized PCNa. Thus, the PCN-inducible family of rat liver cytochrome P-450 comprises at least two separate proteins.     

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2)

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.     

Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli

Cytochrome P-450cam catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exohydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P-450cam structural gene has been cloned by mutant complementation in P. putida (Koga, H., Rauchfuss, B., and Gunsalus, I. C. (1985) Biochem. Biophys. Res. Commun. 130, 412-417). We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5' and 175 base pairs of 3' flanking sequence. Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein (Haniu, M., Armes, L. G., Yasunobu, K. T., Shastry, B. A., and Gunsalus, I. C. (1982) J. Biol. Chem. 257, 12664-12671), five differences were found. The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414. Other differences were: Gln-274----Glu-276, Ser-359----His-361, and Asn-405----Asp-407. N-terminal amino acid sequence analysis of the cloned cytochrome P-450cam enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemo-protein, with the N-terminal Met removed by processing as found in P. putida. Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P. putida. The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the cytochrome P-450cam gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.     

Microheterogeneity in the major phenobarbital-inducible forms of rabbit liver microsomal cytochrome P-450 as revealed by nucleotide sequencing of cloned cDNAs

We have isolated one full-length cDNA clone, termed pHP1, and a number of clones of shorter insert lengths, tentatively called b14, b46, etc., all encoding phenobarbital- (PB-) inducible forms of rabbit liver microsomal cytochrome P-450, and determined their nucleotide sequences. The polypeptides encoded by these cDNAs can be classified into five types, represented by HP1, b14, b46, b52, and b54, the deduced amino acid sequences of which are more than 95% similar to one another. Amino acid differences among them total 24 positions, which are distributed over the entire sequence, in contrast to the microheterogeneity observed in two PB-inducible rat liver microsomal cytochromes P-450 (P-450b and P-450e). The primary structure deduced for the HP1 protein is 97% similar to that determined for rabbit P-450 LM2 (form 2), which has been purified by Coon and co-workers [van der Hoeven, T. A., Haugen, D. A., & Coon, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569-675; Haugen, D. A., & Coon, M. J. (1976) J. Biol. Chem. 251, 7929-7939] as the major PB-inducible form of rabbit liver microsomal cytochrome P-450. The amino acid sequence of P-450(1), which we have purified as the major PB-inducible rabbit liver cytochrome P-450, was partially determined with the sequence reported for P-450 LM2 as a reference. The two sequences are closely similar to each other, but at least two amino acid differences can be detected between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Amino acid variations of cytochrome P-450 lanosterol 14 alpha-demethylase (CYP51A1) from fluconazoleresistant clinical isolates of Candida albicans

We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.     

Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida

Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.     

Isolation of a new form of cytochrome P-450 with prostaglandin A and fatty acid omega-hydroxylase activities from rabbit kidney cortex microsomes

Two different forms of cytochrome P-450, highly active in the omega-hydroxylation of prostaglandin A, and the omega- and (omega-1)-hydroxylation of fatty acids (P-450ka-1 and P-450ka-2), have been purified from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)-phthalate. On the basis of the peptide map patterns and NH2-terminal amino acid sequence, P-450ka-1 was determined to be a new form of omega-hydroxylase cytochrome P-450, whereas P-450ka-2 is identical to P-450ka reported earlier. The first 20 NH2-terminal amino acid sequence (ALNPTRLPGSLSGLLQVAGL) and (ALSPTRLPGSFSGFLQAAGL) of P-450ka-1 and P-450ka-2 showed 90 and 80% homology with that of the lung prostaglandin omega-hydroxylase, respectively, suggesting that these three cytochromes P-450 are members of the same omega-hydroxylase cytochrome P-450 gene family.     

Modification of the nucleotide cofactor-binding site of cytochrome P-450 reductase to enhance turnover with NADH in Vivo

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP(+) makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP(+). Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k(cat)/K(m)) with NADH. Inhibition by NADP(+) was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.     

Structural details of the human cytochrome P-450c gene

Aryl hydrocarbon hydroxylase activity is most closely associated with cytochrome P-450c in the rat and cytochrome P1-450 in the mouse. The sequence for the orthologous human gene coding for this enzymatic activity has been determined from several sources: cytochrome P-450c isolated from human embryonic DNA [K. Kawajiri, J. Watanabe, O. Gotoh, Y. Tagashiri, K. Sogawa, and Y. Fujii-Kuriyama (1986) Eur. J. Biochem. 139, 219-225], human lymphocytes in our own laboratory, and cytochrome P1-450 isolated from the established human breast carcinoma cell line, MCF-7 [A.K. Jaiswal, F. J. Gonzalez, and D. W. Nebert (1985) Nucleic Acids Res. 13, 4503-4520]. The data from our laboratory agree well with the sequence derived from human embryonic DNA, but differs significantly from that reported for the gene isolated from MCF-7 cells. Among these differences are a 320-bp insert and a 650-bp deletion in intron 1 relative to the sequence derived from the established cell line. We observe two mRNA species that hybridize to cytochrome P-450c probes, one expected at 2.7 kb and an additional 2.0-kb species. Finally, we note additional hybridization bands in 11% of the population examined by Southern blot analysis, representing either a second rare allele or, more likely, a duplication of at least a portion of the cytochrome P-450c gene.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

Comparison of primary structures deduced from cDNA nucleotide sequences for various forms of liver microsomal cytochrome P-450 from phenobarbital-treated rabbits

cDNA clones, termed pHP2, b32-3, b43, and b43-1, encoding cytochromes P-450 that are expressed in the liver of phenobarbital- (PB-) treated rabbits were isolated, and their nucleotide sequences were determined. pHP2 cDNA contains an open reading frame for a 490-residue protein and is a full-length counterpart of pP-450PBc2 [Leighton, J. K., Debrunner-Vossbrinck, B. A., & Kemper, B. (1984) Biochemistry 23, 204-210]. The b32-3 insert has a sequence for a protein whose primary structure is 91% similar to that of progesterone 21-hydroxylase P-450 1, though this cDNA lacks the sequence encoding the amino-terminal 110 residues. The overlapping clones b43 and b43-1 together encode an ethanol-inducible form of cytochrome P-450, though the amino-terminal five or more residues are missing in the composite b43/b43-1 sequence. Northern blot analysis showed that the b43/b43-1 protein is more strongly inducible by polycyclic aromatic hydrocarbons and isosafrole than by PB, in contrast to the case of the HP2 and b32-3 proteins. A comparison of the primary structures of eight forms of cytochrome P-450, including the HP2, b32-3, and b43/b43-1 proteins, that are expressed in the liver of PB-treated rabbits showed that 149 out of 487-492 amino acid residues are conserved in these cytochromes P-450. The eight forms can be assigned to three rabbit cytochrome P-450 gene subfamilies, P450IIB, P450IIC, and P450IIE. It was also shown that the members of the rabbit P450IIC subfamily can be further classified into three subgroups on the basis of their sequence similarity.     

Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells

Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy and ferredoxinsoy (soyB) from Streptomyces griseus

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P-450, designated cytochrome P-450soy. A DNA segment carrying the structural gene encoding P-450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P-450soy was compared with that for P-450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe-4S] ferredoxin (soyB), named ferredoxinsoy, was identified.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts

In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.