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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.

ABSTRACT: 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.

SUBMITTER: Iwasaki M

PROVIDER: S-EPMC1136477 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Project description:CYP 3A10 is a hamster liver cytochrome P-450 (P450) that encodes lithocholic acid 6 beta-hydroxylase, an enzyme that plays an important role in the detoxification of the cholestatic secondary bile acid lithocholate. Western-blot analysis revealed that the expression of CYP 3A10 protein is male-specific in hamster liver microsomes, a finding that is consistent with earlier analysis of CYP 3A10 mRNA. Since it has not been established whether the specificities of bile acid hydroxylase P450s, such as CYP 3A10, are restricted to their anionic bile acid substrates, we investigated the role of CYP 3A10 in the metabolism of a series of neutral steroid hormones using cDNA directed-expression in COS cells. The steroid hormones examined, testosterone, androstenedione and progesterone, were each metabolized by the expressed CYP 3A10, with 6 beta-hydroxylation corresponding to a major activity in all three instances. CYP 3A10-dependent steroid hydroxylation was increased substantially when the microsomes were prepared from COS cells co-transfected with NADPH:P450 reductase cDNA. In this case, the expressed P450 actively catalysed the 6 beta-hydroxylation of testosterone (288 +/- 23 pmol of product formed/min per mg of COS-cell microsomal protein), androstenedione (107 +/- 19 pmol/min per mg) and progesterone (150 +/- 7 pmol/min per mg). Other major CYP 3A10-mediated steroid hydroxylase activities included androstenedione 16 alpha-hydroxylation, progesterone 16 alpha- and 21-hydroxylation, and the formation of several unidentified products. CYP 3A10 exhibited similar Vmax. values for the 6 beta-hydroxylation of androstenedione and lithocholic acid (132 and 164 pmol/min per mg respectively), but metabolized the bile acid with a 3-fold lower Km (25 microM, as against 75 microM for androstenedione). Together, these studies establish that the substrate specificity of the bile acid hydroxylase CYP 3A10 is not restricted to bile acids, and further suggest that CYP 3A10 can play a physiologically important role in the metabolism of two classes of endogenous P450 substrates:steroid hormones and bile acids.

Project description:Previous studies of erythromycin biosynthesis have indicated that a cytochrome P-450 monooxygenase system is responsible for hydroxylation of 6-deoxyerythronolide B to erythronolide B as part of erythromycin biosynthesis in Saccharopolyspora erythraea (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210 1987). The enzyme was previously purified to apparent homogeneity and found to have a catalytic turnover number of approximately 10(-3) min-1. More recently, disruption of a P-450-encoding sequence (eryF) in the region of ermE, the erythromycin resistance gene of S. erythraea, produced a 6-deoxyerythronolide B hydroxylation-deficient mutant (J. M. Weber, J. O. Leung, S. J. Swanson, K. B. Idler, and J. B. McAlpine, Science 252:114-116, 1991). In this study we purified the catalytically active cytochrome P-450 fraction from S. erythraea and found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis that it consists of a major and a minor P-450 species. The gene encoding the major species (orf405) was cloned from genomic DNA and found to be distinct from eryF. Both the orf405 and eryF genes were expressed in Escherichia coli, and the properties of the proteins were compared. Heterologously expressed EryF and Orf405 both reacted with antisera prepared against the 6-deoxyerythronolide B hydroxylase described by Shafiee and Hutchinson (1987), and the EryF polypeptide comigrated with the minor P-450 species from S. erythraea on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. In comparisons of enzymatic activity, EryF hydroxylated a substrate with a turnover number of 53 min-1, whereas Orf405 showed no detectable activity with a 6-deoxyerythronolide B analog. Both enzymes showed weak activity in the O-dealkylation of 7-ethoxycoumarin. We conclude that the previously isolated 6-deoxyerythronolide B hydroxylase was a mixture of two P-450 enzymes and that only the minor form shows 6-deoxyerythronolide B hydroxylase activity.

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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.

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