Publications

• Synthesis of C14-17-hydroxy-11-desoxycorticosterone and 17-hydroxycorticosterone by fractionated extracts of adrenal homogenates.

  PLAGER J.E., SAMUELS L.T.

  Arch Biochem Biophys 42:477-478(1953) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli.

  Arase M., Waterman M.R., Kagawa N.

  Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have ... >> More

  Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies. << Less

  Biochem Biophys Res Commun 344:400-405(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Human Cytochrome P450 21A2, the major steroid 21-hydroxylase: structure of the enzyme-progesterone substrate complex and rate-limiting c-h bond cleavage.

  Pallan P.S., Wang C., Lei L., Yoshimoto F.K., Auchus R.J., Waterman M.R., Guengerich F.P., Egli M.

  Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., ... >> More

  Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. << Less

  J. Biol. Chem. 290:13128-13143(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Hydroxylation of steroids at carbon 21.

  RYAN K.J., ENGEL L.L.

  J Biol Chem 225:103-114(1957) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Studies on the steroid hydroxylation system in adrenal cortex microsomes. Purification and characterization of cytochrome P-450 specific for steroid C-21 hydroxylation.

  Kominami S., Ochi H., Kobayashi Y., Takemori S.

  J Biol Chem 255:3386-3394(1980) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The action of adrenal homogenates on progesterone, 17-hydroxyprogesterone and 21-desoxycortisone.

  DORFMAN R.I., HAYANO M.

  Arch Biochem Biophys 36:237-239(1952) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Molecular cloning and expression of guinea pig cytochrome P450c21 cDNA (steroid 21-hydroxylase) isolated from the adrenals.

  Martineau I., Belanger A., Tchernof A., Tremblay Y.

  In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid ... >> More

  In mammals, the P450c21 enzyme mediates 21-hydroxylase activity by transforming progesterone and 17-hydroxyprogesterone into deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC), respectively. Previous studies have shown that among the adrenal steroid hydroxylase enzymes involved in C19 steroid and glucocorticoid syntheses, P450c21 plays an important role, because it is localized at the key branch between glucocorticoids and C19 steroid production. Its implication in congenital adrenal hyperplasia is also of great clinical interest. In this study, in addition to describing the isolation of the P450c21 cDNA from guinea pig (GP) adrenal and comparing it to those from other species, we report on its tissue-distribution and on the activity of the recombinant protein towards progesterone and 17-hydroxyprogesterone. The guinea pig P450c21 includes the full-length coding region (1464 nucleotide) that is translated to a protein of 488 amino acids. The clone shares highly conserved regions with other species. The guinea pig P450c21 cDNA hybridized with a major transcript of 2.1kb and with two minor related transcripts of 1.8 and 1.5 kb and was found to be adrenal-specific among the various tissues analyzed. Characterization of the enzymatic activity by transient transfection of the guinea pig P450c21 cDNA in human embryonic kidney 293 cells indicated a net preference for the 21-hydroxylation of 17-hydroxyprogesterone in comparison to the progesterone substrate. Assays showed a maximum conversion rate of 12.5% for the conversion of progesterone into deoxycorticosterone (mineralocorticoid pathway), whereas the guinea pig P450c21 demonstrated a higher activity with 17alpha-hydroxyprogesterone, with 55% of 11-deoxycortisol formation (glucocorticoid pathway) after 48 h. Adrenocorticotropin and an analogue of the second messenger cyclic adenosine monophosphate specifically increased the abundance of P450c21 mRNA levels in guinea pig adrenal cells. << Less

  J Steroid Biochem Mol Biol 86:123-132(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.