The status of oxygen atoms in the removal of C-19 in oestrogen biosynthesis

Akhtar, M.; Calder, Michael R.; Corina, David L.; Wright, James A.

doi:10.1039/c39810000129

Cited by 43 publications

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“…The incorporation of one atom of 18 O label from O 2 into formic acid (Fig. 3A) had been considered one of the most critical pieces of evidence in support of an FeO 2 Ϫ mechanism for P450 19A1 (14,15,34). Because of the importance of this evidence in the existing dogma, we re-examined this experiment using several technical improvements including the following: (i) purified recombinant P450 19A1; (ii) a new diazo reagent with a pyridine nitrogen to facilitate positive ionization for liquid chromatography-mass spectrometry (LC-MS); and (iii) the use of high resolution mass spectrometry (HRMS) (33).…”

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confidence: 99%

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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We propose three mechanisms that can involve the FeO 3؉ entity and that explain the 18 O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17␣-hydroxylated products to yield 16,17␣-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6␤-hydroxylation of 16␣,17␣-dihydroxyprogesterone and the oxidation of both 16␣,17␣-dihydroxyprogesterone and 16␣,17␣-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17␣,20-lyase reaction cannot be excluded. Cytochrome P450 (P450)3 enzymes catalyze oxidations of more chemicals than any other group of proteins (1). The list of reactions includes aliphatic and aromatic hydroxylations, heteroatom oxidations, epoxidations, and reactions involving both ring formation and cleavage (2-4). Many P450 reactions are important in the biosynthesis and degradation of steroids and sterols (4, 5), including several critical C-C bond cleavage reactions, i.e. those catalyzed by P450s 11A1, 17A1 (Fig. 1), 19A1, and 51A1 (6, 7).The mechanisms of the C-C cleavage reactions have been the subject of considerable interest and debate. One of the questions with P450s 17A1, 19A1, and 51A1 has been whether the active oxidant is a ferric peroxide (FeO 2 Ϫ ), which is an early intermediate following oxygen addition to the iron (Fig. 2, step 4) or the FeO 3ϩ species (Fig. 2, step 6), often referred to as compound I (4, 10, 11). With P450s 17A1 and 19A1, a variety of approaches has been applied, including theoretical calculations, biomimetic models, spectroscopy, substrate atom labeling, and kinetics (12-32).These C-C bond cleavage reactions are complex, and many of the results are ambiguous; also, a "mixed" mechanism would not be discerned in many of these experiments. One powerful approach originally used by Akhtar and co-workers (27-31) analyzes the actual reaction and can provide discrimination between the nucleophilic FeO 2 Ϫ and electrophilic FeO 3ϩ reactions (Fig. 2), based on the incorporation of 18 O label from O 2 into the carboxylic acid products (Fig. 3) (7). However, these experiments are complicated due to the ubiquitous presence of formic acid (P450 19A1 and 51A1 reactions) and acetic acid (P450 17A1) in laboratory settings. Thus, the data from such experiments are interpreted with the most confidence when the steroid substrates are labeled with 2 H or 13 C isotopes to facilitate analysis (15,33). Even then, the mass spectrometry results can be problematic, particularly if a shift of only one atomic mass unit is introduced and isotopologues derived from 18 O incorporation are not discriminated from molecules containing natural abundance 13 C atoms (33). The incorporation of one atom of 18 O label from O 2 into formic acid (F...

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confidence: 99%

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Yoshimoto

Gonzalez

Auchus

et al. 2016

Journal of Biological Chemistry

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“…The resulting adduct, 11, decomposes to furnish the side chain as acetic acid and the ketone functionality at C-17 (17 → 18, Scheme 3). The role for a nucleophilic iron-peroxide anion intermediate (8) in acyl-carbon cleavage was originally highlighted by our studies on aromatase (CYP19). It was found that the third reaction catalysed by aromatase, in the biosynthesis of oestrogens, is attended by the incorporation of an atom of oxygen from O 2 into the released formic acid [9,53].…”

Section: Discussionmentioning

confidence: 98%

“…The dual functionality was originally highlighted by our studies on aromatase (CYP19), which is responsible for the construction, from the androgen nucleus, of the aromatic ring A of the female hormones, oestrone and oestradiol (Scheme 1) [5][6][7][8][9]. The oxidative target for aromatase is the C-19 of androgens (1), which is hydroxylated to produce the alcohol (2), the latter is then converted into the 19-aldehyde (3), presumably via the intermediary of a gem diol.…”

Section: Introductionmentioning

confidence: 98%

The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities

Lee-Robichaud

Akhtar

Wright

et al. 2004

The Journal of Steroid Biochemistry and Molecular Biology

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“…This enzyme is also of interest to a number of investigators (e.g., refs. [6][7][8] because of the complexity of the reaction it catalyzes. Although it has been shown that three molecules of molecular oxygen and six reducing equivalents ofNADPH are consumed during estrogen formation (9), the reaction mechanism is not yet completely understood.…”

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Structure-function studies of human aromatase by site-directed mutagenesis: kinetic properties of mutants Pro-308----Phe, Tyr-361----Phe, Tyr-361----Leu, and Phe-406----Arg.

Zhou

Pompon

Chen

1991

Proc. Natl. Acad. Sci. U.S.A.

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Aromatase, a cytochrome P450, catalyzes the formation of aromatic C-18 estrogenic steroids from C-19 androgens. Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [13, (2)(3)(4)(5) and the inhibition ofthe enzyme as part ofa therapeutic approach to this disease make the study of aromatase of paramount importance. This enzyme is also of interest to a number of investigators (e.g., refs. 6-8) because of the complexity of the reaction it catalyzes. Although it has been shown that three molecules of molecular oxygen and six reducing equivalents ofNADPH are consumed during estrogen formation (9), the reaction mechanism is not yet completely understood. Site-directed mutagenesis may help to unravel the reaction mechanism of aromatase and provide structural information useful in the design of more effective aromatase inhibitor(s) for the treatment of breast cancer.Although the human placental aromatase cDNA has been cloned and the complete amino acid sequence of the enzyme has been deduced from the nucleotide sequence (10-13), the structure-function relationship of the enzyme has yet to be established. A mammalian cell expression plasmid, pH

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The status of oxygen atoms in the removal of C-19 in oestrogen biosynthesis

Abstract: Three 0,-dependent reactions are involved in the removal of C-19 as formate, in oestrogen biosynthesis; it is shown that the oxygen atoms introduced in steps 1 and 3 of the process are the ones which are found in the biosynthetic formate.

Cited by 43 publications

References 0 publications

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities

Structure-function studies of human aromatase by site-directed mutagenesis: kinetic properties of mutants Pro-308----Phe, Tyr-361----Phe, Tyr-361----Leu, and Phe-406----Arg.

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