Characterization and regulation of sex-specific mouse steroid hydroxylase genes

1990 ◽  
Vol 68 (6) ◽  
pp. 754-761 ◽  
Author(s):  
Hidefumi Yoshioka ◽  
Raija Lindberg ◽  
Garry Wong ◽  
Takeshi Ichikawa ◽  
Takao Itakura ◽  
...  
Keyword(s):  
Gene Families ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Hydroxylase Activity ◽  
Steroid Hydroxylase ◽  
Selective Mutation ◽  
Female Specific ◽  
Male Specific ◽  
Cytochrome P 450

We characterized the genes of the male-specific mouse steroid 16α-hydroxylase (C-P-45016α) and the female-specific mouse steroid 15α-hydroxylase (P-45015α) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15α-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.Key words: cytochrome P-450, steroid hydroxylase, site-directed mutagenesis, evolution, gene regulation.

scholarly journals Site-directed mutagenesis of mouse steroid 7α-hydroxylase (cytochrome P-4507α): role of residue-209 in determining steroid-cytochrome P-450 interaction

1993 ◽  
Vol 291 (2) ◽  
pp. 569-573 ◽  
Author(s):  
M Iwasaki ◽  
R L P Lindberg ◽  
R O Juvonen ◽  
M Negishi
Keyword(s):  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Hydroxylase Activity ◽  
Steroid Hydroxylase ◽  
Kd Values ◽  
Important Residue ◽  
Steroid Binding ◽  
Cytochrome P 450

We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae. Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4). The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone. The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable. The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu. The mutant P450(7) alpha displays a 17-fold lower Vmax. value than the wild-type enzyme. Unexpectedly, it also has 3-fold lower Km and Kd values. Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket. Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha. Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold. Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha.

scholarly journals Inhibition of cholesterol 7α-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC

1989 ◽  
Vol 262 (1) ◽  
pp. 91-95 ◽  
Author(s):  
E R Eldredge ◽  
B Jackson ◽  
K E Suckling ◽  
C R Wolf
Keyword(s):  
Bile Acid ◽  
Feedback Regulation ◽  
Structural Similarity ◽  
Gene Families ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Post Translational Modification ◽  
Male Specific ◽  
Cytochrome P 450 ◽  
Gene Subfamily

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.

A dual role of growth hormone as a feminizing and masculinizing factor in the control of sex-specific cytochrome P-450 isozymes in rat liver

1989 ◽  
Vol 120 (2) ◽  
pp. 311-317 ◽  
Author(s):  
A. Mode ◽  
E. Wiersma-Larsson ◽  
A. Ström ◽  
P. G. Zaphiropoulos ◽  
J.-Å. Gustafsson
Keyword(s):  
Time Course ◽  
Mrna Level ◽  
Simultaneous Treatment ◽  
Full Effect ◽  
Hypophysectomized Rats ◽  
Female Specific ◽  
Male Specific ◽  
Cytochrome P 450 ◽  
Secretory Pattern

ABSTRACT The effects of GH on the major constitutive sex-specific forms of cytochrome P-450 (P-45015β and P-45016α) were studied in hypophysectomized rats at the mRNA level. Time-course experiments were performed with or without simultaneous treatment with thyroxine and cortisol. Intermittent administration of GH, mimicking the male secretory pattern, caused complete masculinization of the male specific P-45016α at a pretranslational level in the absence and presence of thyroxine and cortisol. When GH was administered continuously, mimicking the female secretory pattern, the female specific P-45015β was induced, an effect that was dramatically potentiated by simultaneous treatment with thyroxine and cortisol. A synergistic effect of thyroxine and cortisol at a pretranslational level was demonstrated, although the major potentiating effect could be attributed to thyroxine. Thus it was concluded that GH, depending on its secretory pattern is the sole masculinizing factor for cytochrome P-450, and that it is also a feminizing factor, although this activity requires the synergistic action of thyroid hormones and glucocorticoids to reach its full effect. Journal of Endocrinology (1989) 120, 311–317

scholarly journals Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents

1998 ◽  
Vol 42 (2) ◽  
pp. 241-253 ◽  
Author(s):  
Dominique Sanglard ◽  
Françoise Ischer ◽  
Luc Koymans ◽  
Jacques Bille
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Antifungal Agents ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Single Amino Acid ◽  
Efflux Transporters ◽  
Single Mutation ◽  
Multidrug Efflux ◽  
Cytochrome P 450

ABSTRACT The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance ofC. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression inSaccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different clonedCYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.

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