Biotransformation of sterols: selective cleavage of the side chain

This review elaborates on the recent development of microbial sterol biotransformation systems. Particular emphasis is laid on the new enzymatic approach investigating the cleavage of sterol side chain. New developments in the area of immobilized cell system and use of organic media along with recent reviews on side chain cleavage are discussed.     

Stereochemical aspects of the biosynthesis of the side chain of 9beta, 19-cyclopropyl sterols in maize seedlings treated with tridemorph

9β, 19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulate dramatically in maize (Zea mays L. var LG 11) seedlings treated with Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide (M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). In contrast to the situation in control plants where 24-ethyl sterols predominate largely, 24-methyl sterols were more than 98% of total cyclopropyl sterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the 24-methyl cholesterol of control plants. The presence of two epimers at C-24 has been previously explained by the operation of two routes (M. Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One may proceed via Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the (24-R)-24-methyl epimer. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³-, or a Δ²⁵-sterol intermediate to give the (24-S)-24-methyl epimer. Such intermediates have been searched for in excised Zea mays axes grown aseptically in the presence of Tridemorph and either [5-¹⁴C]mevalonic acid, or [Me-¹⁴C]-l-methionine. Whereas Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-cyclopropyl sterols were found in relatively large amounts, only traces of radioactivity were associated with Δ²⁵-sterols. Gas chromatography/mass spectrometry analysis of the sterols from axes grown in the presence of [Me-²H₃]-l-methionine showed that Δ²⁴⁽²⁸⁾-cyclopropyl sterols contained only two ²H atoms at C-28 as expected and that the 24-methyl pollinastanol fraction contained species with two ²H atoms and no species with three ²H atoms. These results indicate that both (24-R)- and (24-S)-epimers originate from a common Δ²⁴⁽²⁸⁾ precursor. After incubation of the axis with [5-¹⁴C,(4-R)-4-³H₁]mevalonic acid, the 24-methyl pollinastanol had a ³H:¹⁴C atomic ratio of 4:6 which is consistent with the intermediacy of a Δ²⁴⁽²⁵⁾-sterol. All these data are in accordance with a pathway where Δ²⁴⁽²⁸⁾-cyclopropyl sterols are isomerized to give Δ²⁴⁽²⁵⁾-cyclopropyl sterols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible mechanism for the reduction step is discussed.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

The biosynthesis of sterols in higher plants

1. [2-(14)C]Mevalonate was incorporated into squalene and the major phytosterols of pea and maize leaves; it was also incorporated into compounds belonging to the 4,4-dimethyl and 4alpha-methyl steroid groups and which may be possible phytosterol intermediates. 2. l-[Me-(14)C]Methionine was incorporated into the major sterols and also into the 4,4-dimethyl and 4alpha-methyl steroid groups. No radioactivity was detected in squalene. 3. Under anaerobic conditions incorporation of [2-(14)C]-mevalonate into the non-saponifiable lipid of pea leaves was drastically decreased but radioactive squalene was accumulated. 4. Cycloartenol, 24-methylenecycloartanol, 24-methylenelophenol, 24-ethylidenelophenol, fucosterol, beta-sitosterol, stigmasterol and campesterol have been identified by gas-liquid chromatography in pea leaves. 5. The significance of these results in connexion with phytosterol biosynthesis and the introduction of the alkyl group at C-24 into phytosterols is discussed.     

Dinosterol side chain biosynthesis in a marine dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinofiagellate, Crypthecodinium cohnii, cultured in a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly synthesized 4α-monomethyl compound dinosterol (4α,23,24-trimethylcholest-22-en-3β-ol). The MS fragmentation pattern indicated that the C-23 methyl group contained three deuterium atoms and was introduced intact by transmethylation from methionine. The C-24 methyl group contained only two deuterium atoms which is consistent with the production of a 24-methylenesterol intermediate which is subsequently reduced to give the 24-methyl side chain. Mechanisms are proposed to account for the production of the dinosterol side chain.     

Synthesis of sterols with modified side chains.

Synthesis of sterols with side chain containing from four to nine carbons are described.     

On the mechanism of side chain oxidation of N-beta-alanyldopamine by cuticular enzymes from Sarcophaga bullata

The metabolism of N-beta-alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N-beta-alanyl-norepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N-acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD-quinone-N-acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea--a well-known phenoloxidase inhibitor--completely inhibited the NBANE production even at 5 microM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as beta-methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II-(Nar-[alpha-3-aminopropionyl amino methyl-3,4-dihydroxybenzyl]-L-kynurenine) could be isolated as by-products, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael-1,6-addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.     

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-(14)C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (alpha-p-chlorophenoxyisobutyrate) and Clofenapate [alpha,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.     

Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells

Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.     

Myxomycete biodiversity of the Colorado Plateau

A rapid biodiversity assessment for myxomycetes (plasmodial slime moulds) was carried out during a two-week field trip to the Colorado Plateau (western U.S.A.). Due to the very arid climate of the region, the moist chamber culture technique formed a major part of the survey. A total of 1165 records belonging to 93 species and 1 variety from 27 genera was collected from the 433 moist chamber cultures prepared with the bark surface of living plants, litter and weathered dung of herbivorous animals. Only 31 specimens of 16 wood-inhabiting species were collected in the field, mainly at higher elevations. Subsequent moist chamber cultures produced numerous bark-inhabiting species that are usually considered to be rare, including Echinostelium coelocephalum, Protophysarum phloiogenum, and Macbrideola declinata. The most common litter-inhabiting species were Didymium mexicanum and Badhamia melanospora (mainly on decaying parts of Opuntia spp. and Agave spp.); the most abundant coprophilous species were Badhamia cf. apiculospora, Fuligo cinerea, Perichaena liceoides, and Licea tenera. Both, species richness and diversity, increase from sagebrush desert to pine-juniper open woodland and pine-oak woodland. The myxomycete biota of the Colorado Plateau displays a high level of similarity to those of other arid regions of the world (mean coefficient of community C_s = 0.67), differing considerably from temperate (mean C_s = 0.34), boreal (mean C_s = 0.49) and tropical biotas (mean C_s = 0.42).     

Inhibition of sterol biosynthesis in animal cells by 14 alpha-alkyl-substituted 15-oxygenated sterols

Reported herein are the results of investigations of the effects of a number of 14 alpha-alkyl-substituted 15-oxygenated sterols, prepared by chemical synthesis, on sterol biosynthesis and the levels of 3-hydroxy-3-methylglutaryl CoA reductase activity in L cells and in primary cultures of fetal mouse liver cells grown in serum-free media. Several of the compounds, most notably 14 alpha-ethyl-5 alpha-cholest-7-en-3 beta, 15 alpha-diol and 14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, were found to be extraordinarily potent inhibitors of sterol synthesis in these cells. For example, the latter compound caused a 50% inhibition of the incorporation of labeled acetate into digitonin-precipitable sterols in L cells in culture at a concentration of 6 X 10(-9) M.     

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain. We have analyzed the epimeric purity of 4-methylsulfinylbutylglucosinolate by NMR methods using a chiral lanthanide shift reagent. The absolute configuration of the sulfoxide group has been established by comparing the ¹H NMR spectra of the two sulfoximine diastereomers of natural 4-methylsulfinylbutylglucosinolate. According to our data, 4-methylsulfinylbutylglucosinolate isolated from broccoli and A. thaliana is a pure epimer and its sulfoxide group has the R_S configuration. The product of the A. thaliana flavin monooxygenase has these same properties demonstrating that the enzyme is stereospecific and supporting its involvement in glucosinolate side chain formation.     

Further studies on the inhibition of sterol biosynthesis in animal cells by 15-oxygenated sterols

The chemical syntheses of a number of C27 15-oxygenated sterols and their derivatives have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in animal cells in culture. Described herein are chemical syntheses of 3 alpha-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en-15-one-3 alpha-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-3 alpha-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en3,7,15-trione, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha, 14 alpha-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 alpha-cholestan-3 beta, 15 alpha-diol, 5 alpha, 14 alpha-cholestan-15 alpha-ol-3-one, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol, 5 alpha, 14 alpha-cholestan-3,15-dione, and 5 alpha, 14 beta-cholestan-3,5-dione. The effects of 8 of the above compounds and of 5 alpha-cholesta-6,8(14)-dien-3 beta-ol-15-one, 3 beta-he misuccinoyloxy-5 alpha-cholest-8(14)-en-15 one, 3 beta-hexadecanoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3,15-dione, 5 alpha-cholesta-6,8(14)-dien-3,15-dione, 5 alpha-cholest-8-en-3 beta, 15 alpha-diol, 5 alpha-cholest-7-en-3 beta, 15 alpha-diol, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha-cholest-8-en-15 alpha-ol-3-one, and 5 alpha-cholest-7-en-15 alpha-ol-3-one on the synthesis of digitonin-precipitable sterols and on levels of HMG-CoA reductase activity have been investigated and compared with previously published data on 7 other C27 15-oxygenated sterols.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.