Publications

• The role of fumarate and TPN in steroid enzymic 11beta-hydroxylation.

  GRANT J.K., BROWNIE A.C.

  Biochim Biophys Acta 18:433-434(1955) [PubMed] [EuropePMC]

• Structural insights into aldosterone synthase substrate specificity and targeted inhibition.

  Strushkevich N., Gilep A.A., Shen L., Arrowsmith C.H., Edwards A.M., Usanov S.A., Park H.W.

  Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart fa ... >> More

  Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11β-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents. << Less

  Mol. Endocrinol. 27:315-324(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Studies on the nature of steroid 11-beta hydroxylation.

  TOMKINS G.M., MICHAEL P.J., CURRAN J.F.

  Biochim Biophys Acta 23:655-656(1957) [PubMed] [EuropePMC]

• The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-, 18-hydroxylation, and aldehyde synthesis.

  Yanagibashi K., Haniu M., Shively J.E., Shen W.H., Hall P.

  In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa ... >> More

  In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum. << Less

  J Biol Chem 261:3556-3562(1986) [PubMed] [EuropePMC]

• On the mechanism of the C11 beta-hydroxylation of steroids.

  HAYANO M., DORFMAN R.I.

  J Biol Chem 211:227-235(1954) [PubMed] [EuropePMC]

• Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli.

  Zoellner A., Kagawa N., Waterman M.R., Nonaka Y., Takio K., Shiro Y., Hannemann F., Bernhardt R.

  The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-contain ... >> More

  The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1. << Less

  FEBS J. 275:799-810(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Modulation of steroid hydroxylase activity in stably transfected V79MZh11B1 and V79MZh11B2 cells by PKC and PKD inhibitors.

  Bureik M., Zeeh A., Bernhardt R.

  We recently observed that treatment of CYP11B2-expressing COS-1 cells with the broad range kinase inhibitor, staurosporine (STS), strongly inhibited aldosterone biosynthesis, indicating that the activity of a kinase might be a prerequisite for steroid hydroxylase activity. In an attempt to identif ... >> More

  We recently observed that treatment of CYP11B2-expressing COS-1 cells with the broad range kinase inhibitor, staurosporine (STS), strongly inhibited aldosterone biosynthesis, indicating that the activity of a kinase might be a prerequisite for steroid hydroxylase activity. In an attempt to identify such kinases, we measured conversion of 11-deoxycortisol (RSS) and 11-deoxycorticosterone (DOC) by V79MZh11B1 and V79MZh11B2 cells, respectively, in the presence of STS and also after treatment with the kinase inhibitors chelerythrine, rottlerin and Gö 6976. The conversion of both substrates by both cell lines was affected in a selective manner by the kinase inhibitors, suggesting that the activity of the novel PKC-delta and either of conventional PKCs or of PKD alter steroid hydroxylation activity, with their influence depending on both the cytochrome P450 tested and on its steroid substrate. << Less

  Endocr. Res. 28:351-355(2002) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.