Biodiversity of CYP51 in trypanosomes.

Sterol 14alpha-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom. They catalyse a single three-step reaction included in all sterol biosynthetic pathways. Plant CYP51s have strict preference towards their physiological substrate O (obtusifoliol), which is C-4-monomethylated. Natural substrates of animal/fungal CYP51 (lanosterol, 24,25-dihydrolanosterol or 24-methylenelanosterol) are C-4-dimethylated. CYP51 from the pathogenic protozoa TB (Trypanosoma brucei) is the first example of O-specific sterol 14alpha-demethylase in non-photosynthetic organisms. Surprisingly, at 83% amino acid identity to the TB orthologue, CYP51 from TC (Trypanosoma cruzi) clearly prefers C-4-dimethylated sterols. Replacement of animal/fungi-like Ile(105) in the B' helix of TC CYP51 with phenylalanine, the residue found in this position in all plant and other trypanosome CYP51s, dramatically increases the ability of the enzyme to metabolize O, converting it into a more plant-like sterol 14alpha-demethylase. A more than 100-fold increase in binding and turnover is observed for the 24-desmethyl analogue of O [N (norlanosterol)], which is found in vivo in procyclic forms of TB and is a good TB CYP51 substrate in vitro. We believe that (i) N is a non-conventional CYP51 substrate, preferred in TB and perhaps other Trypanosomatidae and (ii) functional similarity of TC CYP51 to animal/fungal orthologues is a result of evolutionary convergence (including F105I mutation), leading to different pathways for sterol production in TC versus TB.

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site.

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Delta24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

Soybean sterol composition and utilization by Phytophthora sojae.

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.

Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis.

Sterol 14 alpha-demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 alpha-demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha-demethylase was examined. Like eukaryotic forms, a 3 beta-hydroxy group and a 14 alpha-methyl group are essential for substrate acceptability by the bacterial 14 alpha-demethylase. The high affinity binding of 31-norcycloartenol without detectable activity indicates that the Delta(8)-bond is required for activity but not for binding. As for plant 14 alpha-demethylases, 31-nor-sterols show a binding preference for MT 14DM. Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha-demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha-demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 alpha-demethylase studied so far. - Bellamine, A., A. T. Mangla, A. L. Dennis, W. D. Nes, and M. R. Waterman. Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis. J. Lipid Res. 2001. 42: 128;-136.

Sterol methyl transferase: enzymology and inhibition.

Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine: Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products. SMTs occur generally in nature, but do not occur in animal systems, suggesting that the difference in sterol synthetic pathways can be exploited therapeutically and in insect-plant interactions. The SMT genes from several plants and fungi have been cloned, sequenced and expressed in bacteria or yeast and bioengineered into tobacco or tomato plants. These enzymes share significant amino acid sequence similarity in the putative sterol and AdoMet binding sites. Investigations of the molecular recognition of sterol fitness and studies with stereospecifically labeled substrates as well as various sterol analogs assayed with native or mutant SMTs from fungi and plants have been carried out recently in our own and other laboratories. These analyses have led to an active-site model, referred to as the 'steric-electric plug' model, which is consistent with a non-covalent mechanism involving the intermediacy of a 24beta-methyl (or ethyl) sterol bound to the ternary complex. Despite the seeming differences between fungal and plant SMT activities the recent data indicate that a distinct SMT or family of SMTs exist in these organisms which bind and transform sterols according to a similar mechanistic plan. Vascular plants have been found to express different complements of C(1)/C(2)-activities in the form of at least three SMT isoforms. This enzyme multiplicity can be a target of regulatory control to affect phytosterol homeostasis in transgenic plants. The state of our current understanding of SMT enzymology and inhibition is presented.

Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition.

The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate delta24(25)-sterol acceptor molecules to delta25(27)-24beta-methyl products stereoselectively. Incubation with pairs of substrates--[2H3-methyl]AdoMet and cycloartenol, and AdoMet and [27-(13)C]lanosterol--followed by 1H and 13C NMR analysis of the isotopically labeled products demonstrated the si-face (beta-face attack) mechanism of C-methylation and the regiospecificity of delta25(27)-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3beta-hydroxyl group, a planar nucleus and a side chain oriented into a 'right-handed' structure (20R-chirality) characteristic of the native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors (Ki for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of magnitude less than the Km for cycloartenol of 28 microM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (Ki = 80 microM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a delta24(25) to delta25(27) 24beta-methyl-SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested.

Sterol methyltransferase 1 controls the level of cholesterol in plants.

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.

Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis.

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.

Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis.

Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

Isolation and characterization of an active-site peptide from a sterol methyl transferase with a mechanism-based inhibitor.

Chemical affinity labeling of pure sterol methyl transferase (SMT) from Saccharomyces cerevisiae using the mechanism-based irreversible inhibitor, [3-3H]26,27-dehydrozymosterol, inhibited the SMT with an apparent Ki of 1.1 microM and k(inact) of 1.52 min(-1). The protein-inhibitor adduct was subjected to cleavage with trypsin and the resulting covalently modified peptide was analyzed by Edman sequencing from the N-terminus. The radiochemically labeled ca. 5.0 kDa peptide fragment of the cleavage mixture was shown to be contiguous through 17 residues to a segment that includes a highly conserved hydrophobic motif (Region I, stretching between T78 and F91) characteristic of SMT enzymes. The results confirm that Region I is the sterol binding/active site.

Overexpression, purification, and stereochemical studies of the recombinant (S)-adenosyl-L-methionine: delta 24(25)- to delta 24(28)-sterol methyl transferase enzyme from Saccharomyces cerevisiae.

The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter. This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography. N-Terminal sequence analysis of the first 10 amino acids of the purified SMT protein confirmed the identity of the start triplet and expected primary structure. The enzyme exhibited a turnover number of 0.01/s and an isoelectric point of 5.95. A combination of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric. The purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits. Studies on product formation using [27-13C]zymosterol and [2H3-methyl]AdoMet incubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the original 24,25- double bond. Deduced amino acid sequence comparisons of the SMT polypeptide from S. cerevisiae with related sterol methyl transferase enzymes of plant and fungal origin indicate that there is a significant degree of similarity between these enzymes. Specifically, there is a conserved sequence (in yeast from amino acids ca. 79 to 92 which contains an YEXGWG motif; referred to as Region I) that is not present in other AdoMet-dependent methyl transferase enzymes.

4,4,14 alpha-trimethyl 9 beta,19-cyclo-5 alpha-26-homocholesta-24,26-dien-3 beta-ol: a potent mechanism-based inactivator of delta 24(25)- to delta 25(27)-sterol methyl transferase.

The title compound (4A) was synthesized and tested as a mechanism-based inactivator of the sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii. Using cycloartenol as substrate, 4A was found to exhibit time-dependent inactivation kinetics, generating a Ki value of 30 microM and Kinact value of 0.30 min-1.

Substrate-based inhibitors of the (S)-adenosyl-L-methionine:delta24(25)- to delta24(28)-sterol methyl transferase from Saccharomyces cerevisiae.

A series of 31 side-chain-modified analogs of cholesterol, zymosterol, lanosterol, and cycloartenol and the steroidal alkaloids solasodine and solanidine were studied as inhibitors of (S)-adenosyl-L-methionine:delta24(25)-sterol methyl transferase (SMT) enzyme activity from Saccharomyces cerevisiae. Two classes of sterol methylation inhibitors were tested: substrate analogs, including mechanism-based inhibitors, and transition state analogs. Several novel sterol methylation inhibitors that contained an aza, aziridine, or ammonium group in the sterol side chain were prepared and tested for the first time. The degree and kinetic pattern of methylation inhibition were found to be influenced by the position and nature of the variant functional group introduced into the side chain. The most potent inhibitors of SMT enzyme activity were transition state analog inhibitors (Ki values of 5 to 10 nM) that mimicked the structure and conformation of the natural substrate presumed to form in the ternary complex generated in the transition state. Steroidal alkaloids were potent competitive inhibitors with Ki values ranging from 2 to 30 microM, which is about the Kmapp of zymosterol, ca. 27 microM. An isosteric analog of the natural substrate, zymosterol, in which the 26/27-gem-dimethyl groups were joined to form a cyclopropylidene function is shown to be a potent irreversible mechanism-based inactivator of SMT enzyme activity that exhibits competitive-type inhibition, Ki 48 microM with a K(inact) of 1.52 min(-1). Mechanistic implications of these results provide new insights into the topology of the ternary complex involving sterol-AdoMet-enzyme.

Antifungal sterol biosynthesis inhibitors.

During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach. Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S. cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful. Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors. With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity.

Sterol utilization and metabolism by Heliothis zea.

Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvae did not develop on a sterol-less medium. delta 5-Sterols with a hydrogen atom, a methylene group, an E- or Z-ethylidene group, or an alpha- or beta-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and delta 5-sterols doubly substituted in the side chain at C-24 with an alpha-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3 beta-cholestanol with a 9 beta, 19-cyclopropyl group, gem dimethyl group at C-4, a delta 5,7-bond or delta 8-bond, or by side chain modified sterols that possessed a delta 25(27)-24 beta-ethyl group, delta 23(24)-24-methyl group or 24-ethyl group, or delta 24(25)-24-methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae) treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 micrograms per insect, whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 micrograms per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24-dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).

Sterol specificity of the Saccharomyces cerevisiae ERG6 gene product expressed in Escherichia coli.

The ERG6 gene from Saccharomyces cerevisiae has been functionally expressed in Escherichia coli, for the first time, yielding a protein that catalyzes the bisubstrate transfer reaction whereby the reactive methyl group from (S)-adenosyl-L-methionine is transferred stereoselectively to C-24 of the sterol side chain. The structural requirements of sterol in binding and catalysis were similar to the native protein from S. cerevisiae. Inhibition of biomethylation was observed with fecosterol and ergosterol which suggests that ergosterol may function in wild-type yeast as feedback regulator of sterol biosynthesis.

Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase from Saccharomyces cerevisiae.

The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14 alpha-methyl or 9 beta,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kappa H/kappa D = 1.3. From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated.

Novel sterol transformations promoted by Saccharomyces cerevisiae strain GL7: evidence for 9 beta, 19-cyclopropyl to 9(11)-isomerization and for 14-demethylation to 8(14)-sterols.

Cultures of Saccharomyces cerevisiae strain GL7 (a sterol auxotroph) were incubated with nonradioactive and tritium-labeled cycloartenol, and the sterol composition of the cells was examined by chemical (GLC, TLC, HPLC, MS, 1H-NMR, and 13C-NMR) and radiotracer techniques. Several novel sterols were isolated from the cells including 14 alpha-methyl ergosta-9(11),24(28)-dien-3 beta-ol, 24 beta-methyl-9 beta,19-cyclopropyl ergost-8(14)-en-3 beta-ol, and 9 beta,19-cyclopropyl ergosta-7(8),24(28)-dien-3 beta-ol. GL7 converted [2-3H]cycloartenol to [2-3H]ergosterol in low yield (1% incorporation), whereas [2-3H]lanosterol was converted to [2-3H]ergosterol in high yield (41% incorporation). The degree of sterol absorption and transformation by GL7 was dependent on the type and amount of sterol(s) in the growth medium. The results demonstrate for the first time that yeast may transform 9 beta,19-cyclopropyl sterols to 9(11)-sterols and delta 5-sterols and that 14-demethylation of sterols may proceed in GL7 to double bond formation either in the 8(14)-position or in the 14(15)-position.