Corrigendum

Self Cite

Using homogeneous pig and recombinant human CYP17, the mechanism of the acyl-carbon bond fission involved in the direct cleavage of pregnenolone was studied. It was found that the formation of androsta-5,16-dien-3 beta-ol (5,16-diene) and androst-5-ene-3 beta,17 alpha-diol (17 alpha-hydroxyandrogen) from pregnenolone was catalyzed by both the isoforms and that the two conversions were dependent on the presence of cytochrome b5 (cyt b5). 3 beta-Hydroxyandrost-5-ene-17 beta-carbaldehyde (aldehyde), an analogue of the physiological substrate pregnenolone, was handled as a substrate by both isoforms of CYP17. The aldehyde underwent cleavage to produce the 5,16-diene plus the 17 alpha-hydroxyandrogen, at rates approximately 8- and 3-fold higher than any physiological reaction catalyzed, in the absence of cytochrome b5, by the pig and human CYP17 isoforms, respectively. The stereochemistry of the reaction was studied using the aldehyde labeled with 2H at three strategic positions, 16 alpha, 16 beta, and 17 alpha, with incubations performed under both 16O2 and 18O2. The results showed that the formation of the 5,16-diene is attended by the removal of the 16 alpha-hydrogen atom; all three 2H atoms are retained in the formation of 17 alpha-hydroxyandrogen and its 17 alpha-hydroxyl oxygen originates from O2. Irrespective of the nature of the substrate, or the enzymic conditions used, the 5,16-diene and 17 alpha-hydroxyandrogen were produced in similar ratios, suggesting that their genesis is closely linked. Both the compounds may be envisaged to arise from a peroxy adduct that fragments to give a carbon radical that then undergoes either a disproportionation or an oxygen-rebound reaction. The conclusion was supported by isotope-partitioning experiments when the conversion of a mixture of the unlabeled aldehyde and its isotopomer, containing 2H at 16 alpha as well as 16 beta, led to the enrichment of 2H in 17 alpha-hydroxyandrogen. It is suggested that the mechanistic kinship between hydroxylation and olefin formation, revealed by the present study, also applies to conventional hydroxylation and desaturation reactions.

supporting

confidence: 88%

supporting

confidence: 55%

Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-45017.alpha.; 17.alpha.-hydroxylase-17,20-lyase)

Lee-Robichaud¹,

Shyadehi²,

Wright³

et al. 1995

Self Cite

“…There is a growing body of evidence implicating the involvement of a substrate-bound ferric peroxy intermediate in these reactions. Studies by Akhtar's group (Stevenson et al, 1988;Corina et al, 1991;Miller et al, 1991) as well as by our own (Mak & Swinney, 1992) support involvement of a ferric peroxy intermediate in CYP17 oxidations. We identified 17-0-acetoxytestosterone (AT) as a product of CYP17 oxidation of progesterone and proposed that product formation occurred via Baeyer-Villiger rearrangement of the ferric peroxy intermediate.…”

Section: Chemistrymentioning

confidence: 51%

Androgen Formation by Cytochrome P450 CYP17. Solvent Isotope Effect and pL Studies Suggest a Role for Protons in the Regulation of Oxene versus Peroxide Chemistry

Swinney¹,

Mak²

1994

CYP17 catalyzes the cleavage of the C-17 side chain of progesterone to form androstenedione. The two-step reaction involves an initial 17 alpha-hydroxylation catalyzed by oxene chemistry followed by cleavage of the C-17 side chain. We have recently shown that C-17 side-chain cleavage may involve the rearrangement of a peroxy intermediate via a Baeyer-Villiger rearrangement [Mak, A. Y., & Swinney, D. C. (1992) J. Am. Chem. Soc. 114, 8309]. Accordingly, CYP17 is proposed to catalyze oxidations via both oxene and peroxide chemistry. This study was initiated to investigate the possibility that protons may play a determining role in differentiating between the oxene and peroxide chemistries associated with product formation. The pL dependence of the deuterium solvent isotope effects associated with progesterone oxidation to 17 alpha-hydroxyprogesterone and 17-O-acetyltestosterone and 17 alpha-hydroxyprogesterone oxidation to androstenedione was determined in microsomes from pig testes. The formation of 17 alpha-hydroxyprogesterone is assumed to occur via oxene chemistry and the formation of 17-O-acetyltestosterone and androstenedione by peroxide chemistry. The initial rate of progesterone oxidation to 17 alpha-hydroxyprogesterone was associated with a pL-independent inverse solvent isotope effect (Hk/Dk = 0.75-0.95, in 30% DOD), whereas the rate of oxidation to 17-O-acetyltestosterone was associated with a pL-independent positive solvent isotope effect in the presence of 30% DOD (Hk/Dk approximately 2). In contrast, DOD inhibited the formation of androstenedione from 17 alpha-hydroxyprogesterone in a noncompetitive, pL-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

“…A variety of physiologically occurring as well as xenobiotic aldehydes remain to be examined for deformylation by microsomal P450s. It may be noted in this connection that ketones, by formation of peroxy hemiketals, may undergo a cleavage reaction comparable to the aldehyde lyase reaction for which an enzymebound peroxy hemiacetal capable of undergoing concerted or sequential /3-scission has been proposed (Stevenson et al, 1988;Vaz et al, 1991;Miller et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Aromatization of a Bicyclic Steroid Analog, 3-Oxodecalin-4-ene-10-carboxaldehyde, by Liver Microsomal Cytochrome P450 2B4

Vaz¹,

Kessell²,

Coon³

1994

Several purified isoforms of microsomal cytochrome P450 were previously shown in this laboratory to catalyze the oxidative deformylation of a variety of alpha- or beta-branched aldehydes with the production of olefins and formic acid. In the present study, 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC, numbered according to the convention for steroids) was synthesized as a bicyclic analog of the aldehyde that is known to be the terminal intermediate in the enzymatic conversion of androgens to estrogens. ODEC undergoes aromatization in a reconstituted enzyme system containing liver microsomal cytochrome P450 2B4 and NADPH-cytochrome P450 reductase, along with NADPH and phosphatidylcholine, under aerobic conditions. The products, 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and formic acid, were identified by mass spectrometry. The corresponding 10-carbinol does not undergo oxidative aromatization with P450 2B4, and with ODEC as substrate, other microsomal P450 cytochromes are either weakly active (isoforms 2C3 and 3A6) or inactive (isoforms 2E1, 1A2, and 2G1). Cytochrome b5 stimulates the P450 2B4-catalyzed reaction with ODEC about 2.6-fold but has no effect with the other P450s. In two respects the conversion of the bicyclic model compound to HTN with P450 2B4 was shown to be similar to that of the steroid aromatase reaction. Deuterium in the formyl group of ODEC was retained in the formic acid that was produced and isolated as the 4-nitrobenzyl derivative, and with preparations of ODEC containing deuterium in the 1 alpha position or the 1 alpha and 2 alpha positions, it was shown that the desaturation reaction is specific for removal of the 1 beta-hydrogen, thus involving a stereospecific cis elimination of formate. Cytochrome b5 has no effect on the stereospecificity of the reaction.