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

Abstract

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...

Characterization of the guinea pig 3β-hydroxysteroid dehydrogenase/Δ5–Δ4-isomerase expressed in the adrenal gland and gonads

“…These results suggest the paraphyly of the order rodentia, as demonstrated by Bulow and Bernhardt with the analyses of the CYP11B gene family [49]. This phylogenetic tree represents the traditional view of the myomorpha-caviomorpha clade, which has been observed by several studies [51][52][53].…”

“…The placement in the molecular phylogeny of the guinea pig is controversial depending on the source of sequences and the method of phylogenetic tree construction [38,39,43,[49][50][51].…”

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: Quantification of steroid intermediates and end products in H295R cells

“…In addition, we assayed the conversion of cortisol in COS-1 expressing recombinant human CYP17A1, in the absence and presence of cytochrome b 5 , which augments the lyase reaction, and did not detect 11␤OH-A4 after 8 h by UPLC MS/MS analyses (data not shown). While the conversion of A4 to 11␤OH-A4 by CYP11B1 has been demonstrated for non-primate species [52,53], to our knowledge, the 11␤-hydroxylation of A4 by primate CYP11B1 has only been indirectly implied. We therefore assayed the metabolism of A4, deoxycortisol and DOC in COS-1 cells transiently co-transfected with baboon CYP11B1 and human adrenodoxin (ADX).…”

The Multifaceted Mineralocorticoid Receptor

“…The requirement for a separate system to regulate energy and electrolyte homeostasis occurred relatively recently. The consensus is that 11-deoxycorticosterone (DOC), corticosterone, and cortisol were the ligands for MR before the CYP11B2 enzyme required to make aldosterone from DOC evolved from a gene duplication of the DNA of the primordial CYP11B1, the last enzyme in corticosterone and cortisol synthesis, following the divergence of the two receptors (3,21,45,54,134,224,243,345). Some mammals, do not have a separate CYP11B2 gene, among these are cattle from whence aldosterone was first isolated (198).…”