Molecular Cloning and Functional Expression of the Cytochrome P450 11B-Hydroxylase of the Guinea Pig

Bülow, Hannes E.; Möbius, K.; Bähr, V.; Bernhardt, Rita

doi:10.1006/bbrc.1996.0591

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“…A total of 1 × 10 6 clones of a guinea pig genomic library (Stratagene, #946110) were screened under low stringency conditions as described for Southern blots using a guinea pig CYP11B1 full‐length probe (1618 bp Xba I fragment of pHBL 5 [1]. Positive clones were purified to homogeneity and analysed by Southern blotting using various restriction endonucleases.…”

Section: Methodsmentioning

confidence: 99%

“… In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11β‐hydroxylase [Bülow, H.E.,Möbius, K., Bähr, V. & Bernhardt, R. (1996) Biochem. Biophys.…”

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confidence: 99%

“…Accordingly, mineralocorticoids are produced by the outer zona glomerulosa while glucocorticoids are formed in the two inner layers of the cortex, the zonae fasciculata/reticularis . Originating from cholesterol they are synthesized by a number of consecutive oxidations and dehydrogenations where all oxidative reactions are catalysed by enzymes of the cytochrome P450 superfamily [2]. The first and rate‐limiting step is the conversion of cholesterol to pregnenolone by the mitochondrial cytochrome P450 side‐chain cleavage enzyme (P450 scc , CYP11A1).…”

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Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity

Bülow

Bernhardt

2002

European Journal of Biochemistry

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In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11b-hydroxylase [Bu¨low, H.E., Mo¨bius, K., Biochem. Biophys. Res. Commun. 221,[304][305][306][307][308][309][310][311][312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11b-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11b-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals.Keywords: guinea pig; 11b-hydroxylase; aldosterone synthase; phylogeny.Higher vertebrates regulate vital processes like volume/ electrolyte homeostasis and glucose/lipid metabolism by means of steroid hormones, namely mineralocorticoids and glucocorticoids. The biosynthesis of these steroids occurs primarily in the adrenal cortex within morphologically and functionally distinct zones. Accordingly, mineralocorticoids are produced by the outer zona glomerulosa while glucocorticoids are formed in the two inner layers of the cortex, the zonae fasciculata/reticularis. Originating from cholesterol they are synthesized by a number of consecutive oxidations and dehydrogenations where all oxidative reactions are catalysed by enzymes of the cytochrome P450 superfamily [2]. The first and rate-limiting step is the conversion of cholesterol to pregnenolone by the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450 scc , CYP11A1). Subsequently, pregnenolone is dehydrogenated and oxidized in position 17 and/or 21 to yield 11-deoxycortisol or 11-deoxycorticosterone, respectively.Both compounds in turn are substrates for the cytochrome P450 enzymes of the CYP11B subfamiliy, namely the 11b-hydroxylase (CYP11B1) and the aldosterone synthase (CYP11B2). While CYP11B1 hydroxylates 11-deoxycortisol in position 11 to give cortisol as the major glucocorticoid, the clos...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity

Bülow

Bernhardt

2002

European Journal of Biochemistry

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“…However, it cannot convert corticosterone to aldosterone. 1,5 Two isozymes of 11β-hydroxylase have been isolated in mice, 11 rats, 12 and guinea pigs, 13,14 but in cows, 15 pigs, 16 and frogs, 17 a single enzyme is responsible for both glucocorticoid and mineralocorticoid synthesis. 1,5 The CYP11B2 isozyme has weaker activity in converting deoxycorticosterone to corticosterone, but it can convert corticosterone to aldosterone.…”

Section: Small Animalsmentioning

confidence: 99%

Congenital adrenal hyperplasia secondary to 11β-hydroxylase deficiency in a domestic cat

Knighton¹

2004

javma

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A calico-colored domestic shorthair cat was examined because of possible cryptorchidism. The cat had a fully formed penis, prepuce, and scrotum, but no descended testes, and exploratory laparotomy revealed a grossly normal female internal genital tract (ie, 2 ovaries, 2 uterine horns, and uterine body). Chromosomal analysis revealed a normal female (38,XX) karyotype. Four months later, the cat was examined because of polyuria, polydipsia, and inappropriate urination. Serum cortisol and aldosterone concentrations were low, and results of an ACTH stimulation test were suggestive of decreased adrenal gland function. Serum ACTH, testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and deoxycorticosterone concentrations were high, and a diagnosis of congenital adrenal hyperplasia secondary to 11beta-hydroxylase deficiency was made. Treatment with prednisone diminished clinical signs but had a variable effect on corticosteroids hormone concentrations. To the author's knowledge, this is the first report of congenital adrenal hyperplasia in a cat.

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“…Glucocorticoids and adrenal androgens are synthesized in the zona fasciculata/reticularis, whereas aldosterone, the most potent natural mineralocorticoid, is synthesized in zona glomerulosa cells (Miller and Tyrell, 1995). CYP11B enzymes of other species have also been studied, and it turned out that in bovine (Wada et al, 1985), porcine , and frog (Nonaka et al, 1995) adrenal cortex, synthesis of gluco-and mineralocorticoids is catalyzed by a single enzyme, while in man, rat (Matsukawa et al, 1990), mouse (Domalik et al, 1991), and guinea pig (Bulow et al, 1996;Bulow and Bernhardt, 2002) two distinct isoforms are specialized in the formation of either mineralo-or glucocorticoids. The final steps in cortisol and aldosterone synthesis are performed by two very closely related enzymes, namely CYP11B1 and CYP11B2 (Mornet et al, 1989;Kawamoto et al, 1990a,b).…”

Section: Physiological Function Of Cytochrome P450-dependent Steroid mentioning

confidence: 99%

The Human Steroid Hydroxylases CYP11B1 and CYP11B2

Bureik¹,

Lisurek²,

Bernhardt³

2002

Biological Chemistry

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Major advances have been made during the last decade in our understanding of adrenal steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from steroid hormone-related diseases, from mutagenesis studies and from the construction of three-dimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo- and regiospecific steroid hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.

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Molecular Cloning and Functional Expression of the Cytochrome P450 11B-Hydroxylase of the Guinea Pig

Cited by 20 publications

References 0 publications

Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity

Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity

Congenital adrenal hyperplasia secondary to 11β-hydroxylase deficiency in a domestic cat

The Human Steroid Hydroxylases CYP11B1 and CYP11B2

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