The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.     

Is Pneumocystis a Plant?

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.     

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, Delta(5,7) sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form Delta(22) sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen. The lanosterol derivatives, 24-methylenelanost-8-en-3 beta-ol and (Z)-24-ethylidenelanost-8-en-3 beta-ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci, were also detected among the sterols of the rat-derived P. carinii organisms.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

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.     

Pneumocystis carinii sterol 14α-demethylase activity in Saccharomyces cerevisiae erg11 knockout mutant: sterol biochemistry

Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.     

Biochemical research elucidating metabolic pathways in Pneumocystis

Advances in sequencing the Pneumocystis carinii genome have helped identify potential metabolic pathways operative in the organism. Also, data from characterizing the biochemical and physiological nature of these organisms now allow elucidation of metabolic pathways as well as pose new challenges and questions that require additional experiments. These experiments are being performed despite the difficulty in doing experiments directly on this pathogen that has yet to be subcultured indefinitely and produce mass numbers of cells in vitro. This article reviews biochemical approaches that have provided insights into several Pneumocystis metabolic pathways. It focuses on 1) S-adenosyl-L-methionine (AdoMet; SAM), which is a ubiquitous participant in numerous cellular reactions; 2) sterols: focusing on oxidosqualene cyclase that forms lanosterol in P carinii; SAM:sterol C-24 methyltransferase that adds methyl groups at the C-24 position of the sterol side chain; and sterol 14alpha-demethylase that removes a methyl group at the C-14 position of the sterol nucleus; and 3) synthesis of ubiquinone homologs, which play a pivotal role in mitochondrial inner membrane and other cellular membrane electron transport.     

Identification of C31 and C32 Sterols in Pneumocystis carinii hominis-Infected Human Lungs

SUMMARY Two sterols in autopsied whole lung specimens obtained from Pneumocystis carinii pneumonia patients were detected by gas-liquid chromatography and their structures were elucidated by mass spectrometry and nuclear magnetic resonance spectrometry. Both were in the lanosterol series; the C₃₁ sterol, with a methyl group at C-24, was identified as euphorbol, and the more abundant C₃₂ sterol, with an ethyl group at C-24, is given the trivial name pnemocysterol.     

Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase

The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763).     

Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.

In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem. 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. USA 84:4012-4016, 1987). The sterol biosynthetic pathway in S. cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol. ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated. Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S. cerevisiae. However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake. These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity. Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59.     

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.     

Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.     

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.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Enzyme mechanisms for sterol C-methylations

The mechanisms by which sterol methyl transferases (SMT) transform olefins into structurally different C-methylated products are complex, prompting over 50 years of intense research. Recent enzymological studies, together with the latest discoveries in the fossil record, functional analyses and gene cloning, establish new insights into the enzymatic mechanisms of sterol C-methylation and form a basis for understanding regulation and evolution of the sterol pathway. These studies suggest that SMTs, originated shortly after life appeared on planet earth. SMTs, including those which ultimately give rise to 24 alpha- and 24 beta-alkyl sterols, align the si(beta)-face pi-electrons of the Delta(24)-double bond with the S-methyl group of AdoMet relative to a set of deprotonation bases in the active site. From the orientation of the conformationally flexible side chain in the SMT Michaelis complex, it has been found that either a single product is formed or cationic intermediates are partitioned into multiple olefins. The product structure and stereochemistry of SMT action is phylogenetically distinct and physiologically significant. SMTs control phytosterol homeostasis and their activity is subject to feedback regulation by specific sterol inserts in the membrane. A unified conceptual framework has been formulated in the steric-electric plug model that posits SMT substrate acceptability on the generation of single or double 24-alkylated side chains, which is the basis for binding order, stereospecificity and product diversity in this class of AdoMet-dependent methyl transferase enzymes. The focus of this review is the mechanism of the C-methylation process which, as discussed, can be altered by point mutations in the enzyme to direct the shape of sterol structure to optimize function.     

ESR determination of membrane order parameter in yeast sterol mutants.

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.     

A sterol methyltransferase from bean rust uredospores,Uromyces phaseoli

A methyltransferase(s) that catalyzes the transfer of the methyl group from S-adenosylmethionine to a sterol acceptor was solubilized with Triton X-100 and partially purified from bean rust uredospores (Uromyces phaseoli). Zymosterol was the most active substrate tested while desmosterol and lanosterol exhibited good activity. The products were sterols with either a methylene or ethylidene group at the C-24 position. Direct evidence for the synthesis of the ethylidene group was obtained by using 24-methylenecholesterol as a substrate.     

Evidence for the Presence of "Metabolic Sterols" in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii , respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β -sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C₂₈ and C₂₉ sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii , probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own "metabolic sterols"     

Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.