Biosynthesis of Pregnenolone from Cholesterol by Mitochondrial Enzymes of Bovine Adrenal Cortex. The Question of the Participation of the 20(22)-Olefins and 20,22-Epoxides of Cholesterol

Abstract

The two isomeric 20(22)-olefins and the four isomeric 20,22-epoxides of cholesterol have been examined as possible intermediates in the conversion of cholesterol to pregnenolone both by crude extracts of acetone-dried bovine adrenal cortex mitochondria, and by a partially purified cytochrome P-450 specific for the side chain cleavage ( P -4 5 0~~~) .The quantities of pregnenolone (measured by radioimmunoassay) formed from these putative intermediates and from cholesterol, (2057-20-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (20R,22R)-20,22-dihydroxycholesterol have been compared. In both crude and purified systems, the conversion of the monohydroxycholesterols is somewhat more rapid than that of cholesterol. However, the dihydroxycholesterol is a very much more efficient precursor of pregnenolone. Whereas small amounts of pregnenolone were formed from the four 20,22-epoxycholesteroIs, and slightly larger amounts from the two 20(22)-didehydrocholesterols in the crude enzyme systems, almost no conversion of these six compounds was observed with the purified system containing cytochrome P-45Oscc, adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. It is concluded that the olefins and epoxides are not obligatory intermediates in the conversion of cholesterol to pregnenolone, and the conversions of cholesterol olefins and epoxides observed by us and by others may reflect the presence of extraneous enzymes such as double bond reductases and epoxide hydratases which are unrelated to pregnenolone biosynthesis. The relative effectiveness of the cholesterol olefins and epoxides, as well as 20-isocholesterol in inhibiting the formation of pregnenolone from [4-14C]cholesterol has been determined. The compounds which have the same configuration as cholesterol at C-20 are more potent inhibitors of this conversion than the olefins in which the C-21, C-20, C-22, C-23 carbon system is planar, or than those compounds which have the opposite configuration to cholesterol at C-20 (i.e. 20-isocholesterol, (20S,22S)-20,22-epoxycholesterol and (20S,22R)-20,22-epoxycholesterol). The enzyme appears to be more fastidious in its steric requirement at C-20 than at C-22. Since the olefins and epoxides are stereochemically rigidified around the C-20 to C-22 bond, these inhibitor studies suggest that the natural conformation of cholesterol on the side chain cleavage enzyme system is such that the side chain is fully extended and thus closely resembles the conformation of cholesterol in the crystal state. Abbreviations. The following abbreviations are used : cytochrome P-45OSCC, a highly purified bovine adrenal cortex mitochondrial cytochrome P-450 that cleaves the side chain of cholesterol to pregnenolone and is inactive in the 11 P-hydroxylation reaction; pregnenolone, 3a-hydroxypregn-5-en-20-one; cyanoketone, 2cc-cyEnzymes. Adrenodoxin reductase (EC 1.6.7.1); glucose-6-phosphate dehydrogenase (EC 1 .I. 1.49).

Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

“…Whether the side chain is oriented in extended configuration from carbon 23 as proposed by Teicher et al (1978) or is folded back toward the cholesterol nucleus, as we believe, is not demonstrable from these data and, in fact, may not be important for understanding the side-chain cleavage.…”

Cytotoxic Steroidal Alkaloids from Kibatalia laurifolia

“…The structures of the steroidal alkaloids from K. laurifolia suggested that they could be biosynthesized from the same precursor (see Figure S1, Supporting Information). As in the well-known biosynthesis pathway of steroids, compound 10 should be derived from cholesterol ( 9 ) in the same manner as pregnenolone. − Compounds 1 and 4 could then be produced from 10 by an amination process followed by N -methylation to yield compounds 2 and 5 , respectively. On the other hand, acetylation of 4 should provide compound 6 , while compound 7 could be biosynthesized from 5 and 6 by dehydrogenation followed by an acetylation or methylation process, respectively.…”

Muriceanol, a 24(28)‐Epoxide Sterol Link in the Carbon Flux Toward Side‐Chain Dealkylation of Sterols

“…[10] The (22S)-22-hydroxy group of 1 (path a) appears to be an advantageous functionalization for steroidogenic side-chain cleavage when compared to cholesterol since the conversion of 6 proceeds at twice the rate of that of cholesterol. [11] Scheme 1. Possible biogenetic pathways for 1-3.…”