scholarly journals Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209

1995 ◽  
Vol 306 (1) ◽  
pp. 29-33 ◽  
Author(s):  
M Iwasaki ◽  
D G Davis ◽  
T A Darden ◽  
L G Pedersen ◽  
M Negishi
Keyword(s):  
Dynamic Equilibrium ◽  
The Other ◽  
Axial Position ◽  
Hydroxylase Activity ◽  
Steroid Molecule ◽  
Multiple Binding ◽  
Critical Residues ◽  
Steroid Binding ◽  
Binding Orientations ◽  
Cytochrome P 450

The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates. P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209. Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite. On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced. Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209. Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem. Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis. These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.

IN-VITRO METABOLISM OF 4-ANDROSTENE-3,17-DIONE BY HEPATIC MICROSOMES FROM THE RAINBOW TROUT (SALMO GAIRDNERII): EFFECTS OF HYPOPHYSECTOMY AND OESTRADIOL-17β

1981 ◽  
Vol 90 (1) ◽  
pp. 103-112 ◽  
Author(s):  
TIIU HANSSON ◽  
JAN-ÅKE GUSTAFSSON
Keyword(s):  
Rainbow Trout ◽  
Liver Microsomes ◽  
The Other ◽  
Sham Operation ◽  
Hydroxylase Activity ◽  
Oxidoreductase Activity ◽  
Hepatic Microsomes ◽  
Cytochrome P 450 ◽  
Reductive Metabolism

The metabolism of 4-androstene-3,17-dione by liver microsomes from juvenile rainbow trout, Salmo gairdnerii, was studied in vitro. Hypophysectomy of the fish significantly increased mean hepatic 17-hydroxysteroid oxidoreductase activity when compared with that from sham-operated fish but none of the other enzyme activities investigated were affected. Administration of oestradiol-17β resulted in a significant decrease in mean hepatic 6β-hydroxylase activity and total cytochrome P-450 content but had no effect on the 16-hydroxylation or on the reductive metabolism of androstenedione. The effect of oestradiol-17β on hepatic 6β-hydroxylase activity was as pronounced after hypophysectomy as after sham-operation indicating that these effects of oestradiol-17β are mainly direct and independent of the pituitary gland. The results indicate that hypophysial hormone(s) as well as oestradiol-17β play a role in the regulation of hepatic steroid metabolism in trout.

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 Structural alteration of mouse P450coh by mutation of glycine-207 to proline: spin equilibrium, enzyme kinetics, and heat sensitivity

1993 ◽  
Vol 294 (1) ◽  
pp. 31-34 ◽  
Author(s):  
R O Juvonen ◽  
M Iwasaki ◽  
T Sueyoshi ◽  
M Negishi
Keyword(s):  
Water Molecule ◽  
High Spin ◽  
High Spin State ◽  
Structural Alteration ◽  
Axial Position ◽  
Heat Sensitivity ◽  
Hydroxylase Activity ◽  
Coumarin 7 ◽  
Haem Protein ◽  
Cytochrome P 450

Mouse cytochrome P450coh is a high-spin haem protein which specifically catalyses coumarin 7-hydroxylase activity. A mutation of Gly-207 to Pro shifts the P450coh completely to the low-spin form, indicating that the sixth axial position of the haem is hexaco-ordinated with a water molecule in the mutant G207P. Moreover, the G207P mutation increases the Km value for coumarin 7-hydroxylase activity 100-fold and the Kd value for coumarin binding 200-fold. Conversely, the mutation decreases the Ki and Kd values 10- and 20-fold respectively when testosterone, a larger molecule, is used as a substrate. The results, therefore, are consistent with an idea that the substrate pocket may be larger in the mutant G207P than in the wild-type cytochrome P-450. A Gly-207 to Ala mutation (G207A) of P450coh (G207A), on the other hand, affects neither the spectral nor the enzymic properties of P450coh. Pro-207, through cis/trans isomerization or formation of a kink, may confer on the G207P a structural alteration of its substrate-haem pocket. Our previous studies [Iwasaki, Juvonen, Lindberg and Negishi (1991) J. Biol. Chem. 266, 3380-3382; Juvonen, Iwasaki and Negishi (1991) J. Biol. Chem. 266, 16431-16435] show that the residue at position 209 in P450coh resides close to the sixth axial position of the haem, and the spin equilibrium of the cytochrome P-450 shifts toward the high-spin state as residue 209 becomes more hydrophobic and larger. A Gly-207 to Pro mutation, therefore, results in the creation of a larger substrate pocket in the mutant cytochrome P-450 by altering the protein structure around residue 209 so that a water molecule and testosterone can be accommodated.

Enzymatic Synthesis of 4′-and 3′, 4′ -Hydroxylated Flavanones and Flavones with Flower Extracts of Sinningia cardinalis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1193-1199 ◽  
Author(s):  
K. Stich ◽  
G. Forkmann
Keyword(s):  
Enzymatic Synthesis ◽  
Microsomal Fraction ◽  
Chalcone Synthase ◽  
Condensation Reaction ◽  
Flavonoid Biosynthesis ◽  
Hydroxylase Activity ◽  
Key Enzyme ◽  
Flavone Synthase ◽  
Flavone Synthase Ii ◽  
Cytochrome P 450

Flowers of Sinningia (syn. Rechsteineria) cardinalis contain glycosides of the flavones apigenin (4′-OH) and luteolin (3′,4′-OH) respectively, and of the related 3-deoxyanthocyanidins apigeninidin and luteolinidin. Studies on substrate specificity of the key enzyme of flavonoid biosynthesis, chalcone synthase, revealed that the 3′,4′-hydroxylated flavonoids are formed by hydroxylation of flavonoid compounds rather than by incorporation of caffeoyl-CoA into the flavonoid skeleton during the condensation reaction. In fact, flavonoid 3′-hydroxylase activity could be demonstrat­ed in the microsomal fraction of the flower extracts. The enzyme catalyses hydroxylation of naringenin and apigenin in the 3′-position to eriodictyol and luteolin, respectively, with NADPH as cofactor. Besides flavanone 3′-hydroxylase a further NADPH-dependent enzyme activity (flavone synthase II) was observed in the microsomal fraction catalysing the oxidation of naringenin to apigenin and of eriodictyol to luteolin. The Cytochrome P-450 inhibitor ancymidol was found to abolish completely flavone synthase II activity, whereas flavonoid 3′-hydroxylase activity was not impaired.

Molecular Modelling of the Interaction between DCMU and the QB-Binding Site of Photosystem II

1993 ◽  
Vol 48 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Simon P. Mackay ◽  
Patrick J. O ’Malley
Keyword(s):  
Hydrogen Bond ◽  
Photosystem Ii ◽  
Binding Site ◽  
Binding Sites ◽  
The Other ◽  
Reaction Centre ◽  
Protein Model ◽  
Intermolecular Energy ◽  
Herbicide Binding ◽  
Binding Orientations

Abstract The prefered binding orientations for the herbicide DCMU within the QB-binding site of the D 1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to and the other to the Ser 264 residue. In both cases, which are more important for the stabilization of the interactions.

A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

1998 ◽  
Vol 274 (5) ◽  
pp. C1245-C1252 ◽  
Author(s):  
David J. Morris ◽  
Syed A. Latif ◽  
Michael D. Rokaw ◽  
Charles O. Watlington ◽  
John P. Johnson
Keyword(s):  
Glucocorticoid Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mineralocorticoid Receptors ◽  
Hydroxylase Activity ◽  
A6 Cells ◽  
Ru 28318 ◽  
The One ◽  
Cytochrome P 450 ◽  
Significant Increment

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11β-hydroxysteroid dehydroxgenase (11β-HSD1 or 11β-HSD2) enzymatic activity and so are without apparent “protective” enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6β-hydroxylase that transforms corticosterone to 6β-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3α,5β-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11β-HSD1) and 11-dehydrocorticosterone and 11β-hydroxy-3α,5β-tetrahydroprogesterone (inhibitors of 11β-HSD2) and carbenoxalone, which inhibits both 11β-HSD1 and 11β-HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current ( I sc)] in A6 cells. All of these 11β-HSD inhibitory substances induced a significant increment in corticosterone-induced I sc, which was detectable within 2 h. However, none of these agents caused an increase in I sc when incubated by themselves with A6 cells. In all cases, the additional I sc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original I scelicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6β-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6β-hydroxylase inhibition and the MR-mediated increase in I scin the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6β-hydroxylase and also significantly enhanced corticosterone-induced I sc at 2 h. These experiments indicate that the enhanced MR-mediated I sc in A6 cells may be related to inhibition of 6β-hydroxylase activity in these cells and that this 6β-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).

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