Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity

Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C(1)-transfer activity; SMT1) and stigmastane (C(2)-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C(1)-transfer in concerted fashion to form a single product whereas the protozoan and plant SMTs are bifunctional capable of catalyzing two sequential, mechanistically distinct C-methylation activities in the conversion of a Delta(24)-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C(1) or C(2)-transfer activities typical of the protozoan and plant SMT. In this study, scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicated that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C(1)-transfer activity to include plant-like C(2)-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Erg6p regulate the course of the C-methylation reaction toward product differences.     

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 methyltransferase2: purification, properties, and inhibition

Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))相似文献   

Cloning,mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a K_mapp of 38 μM and k_catapp of 0.14 min⁻¹. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-¹³C]- or [24-²H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of ¹H and ¹³CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ²⁴⁽²⁵⁾-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K_i 14 nM) and 26,27-dehydrolanosterol (K_i 54 μM and k_inact of 0.24 min⁻¹) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC₅₀, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C₁-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.     

Sterol composition and growth of transgenic tobacco plants expressing type-1 and type-2 sterol methyltransferases

Transgenic tobacco (Nicotiana tabacum L.) plants with altered sterol composition were generated by transformation with plant cDNAs encoding type-1 and type-2 sterol methyltransferases (SMTs; EC 2.1.1.41). For both SMT1 and SMT2 transformants, the transformation was associated with a reduction in the level of cholesterol, a non-alkylated sterol. In SMT1 transformants a corresponding increase of alkylated sterols, mainly 24-methyl cholesterol, was observed. On the other hand, in SMT2 transformants the level of 24-methyl cholesterol was reduced, whereas the level of sitosterol was raised. No appreciable alteration of total sterol content was observed for either genotype. The general phenotype of transformants was similar to that of controls, although SMT2 transformants displayed a reduced height at anthesis. The results show that plant sterol composition can be altered by transformation with an SMT1 cDNA without adverse effects on growth and development, and provide evidence, in planta, that SMT1 acts at the initial step in sterol alkylation. Received: 27 June 2000 / Accepted: 22 July 2000     

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.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

Sterol C24-methyltransferase: mechanistic studies of the C-methylation reaction with 24-fluorocycloartenol

The mechanism of the C-methylation reaction was studied with the allylic substrate analog 24-fluorocycloartenol 10 assayed with soybean sterol C24-methyltransferase (SMT). 10 is an effective competitive inhibitor (Ki = 32 microM) of the SMT, and the electron-withdrawing alpha-fluorine substituent was shown to suppress the rate of the C-methylation reaction by one order of magnitude relative to the natural cycloartenol substrate, kcat = 0.02 min(-1) versus 0.6 min(-1); alternately 10 can prevent the critical hydride shift of H24 to C25 to afford time-dependent inactivation of SMT (k(inact) = 0.32 min(-1)).     

Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: functional analysis of conserved aromatic amino acids in Region 1

Soybean sterol methyltransferase (SMT) in the presence of AdoMet catalyzes the transmethylation of the delta24-bond of the sterol side chain to produce phytosterols with a methyl(lene) or ethyl(idene) group at C-24. The function of six aromatic amino acids associated with the putative active center of the SMT, i.e., Region 1 that extends from Phe82 to Phe93 in soybean SMT, was studied by site-directed mutagenesis and heterologous expression in BL21(DE3) bacterial cells. The enzyme-generated products were characterized kinetically and by GC-MS analysis. Substitution of the aromatic amino acids at positions 82, 83, 85, 87, 91, and 93 with a leucine residue produced mutant SMTs with varying activities. The mutants converted cycloartenol to 24(28)-methylene cycloartanol [C1-activity] from a few percent to as much as 95% of the control activity. In contrast, none of the leucine mutants were found to catalyze 24(28)-methylene lophenol [C2-activity], suggesting a loss of function associated with the second C1-transfer activity. In contrast to the loss of the second C1-transfer activity of the Phe82Leu, replacement of the Phe82 residue to isoleucine had minimal effect on the first or second C1-transfer activities, suggesting that the increased bulk (branching) in the leucine side chain contributes to significant perturbations in the active site that generate inaccurate positioning of the substrate side chain disfavoring the second C1-transfer activity. Replacement of Tyr83 to phenylalanine resulted in an increase of the specificity constant (kcat/Km) for the substrate of the second C1-transfer activity by a factor of 5 compared to control and an increase of delta24(28)Z-ethylidene sterol formation in the 24-ethyl sterol product set, suggesting that loss of steric bulk from the phenolic hydroxyl group on tyrosine generates a less precise fit of the delta24(28) sterol side chain into the active site favoring the second C1-transfer activity and prompting reaction channeling during catalysis. Circular dichroism spectra, equilibrium dialysis studies of AdoMet, and chromatographic information of the wild-type and Tyr83 mutants confirmed retention of the overall conformation of the enzyme during the experiments. Together, these findings suggest that the amino acids of Region 1 provide a tight substrate orientation imposed by hydrophobic interactions between the sterol side chain and the SMT active site contacts and control the production and processing of the transmethylation pathways governed by the first and second C1-transfer activities.     

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GAL4 regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.     

Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei

Sterol methyltransferase (SMT) plays a key role in sterol biosynthesis in different pathogenic organisms by setting the pattern of the side chain structure of the final product. This catalyst, absent in humans, provides critical pathway-specific enzymatic steps in the production of ergosterol in fungi or phytosterols in plants. The new SMT gene was isolated from Trypanosoma brucei genomic DNA and cloned into an Escherichia coli expression system. The recombinant SMT was purified to homogeneity to give a band at 40.0 kDa upon SDS-PAGE and showed a tetrameric subunit organization by gel chromatography. It has a pH optimum of 7.5, an apparent kcat value of 0.01 s(-1), and a Km of 47 +/- 4 microm for zymosterol. The products of the reaction were a mixture of C24-monoalkylated sterols, ergosta-8,24 (25)-dienol, ergosta-8,25 (27)-dienol, and ergosta-8,24 (28)-dienol (fecosterol), and an unusual double C24-alkylated sterol, 24,24-dimethyl ergosta-8,25 (27)-dienol, typically found in plants. Inhibitory profile studies with 25-azalanosterol (Ki value of 39 nm) or 24(R,S), 25-epiminolanosterol (Ki value of 49 nm), ergosterol (Ki value of 27 microm) and 26,27-dehydrozymosterol (Ki and kinact values of 29 microm and 0.26 min(-1), respectively) and data showing zymosterol as the preferred acceptor strongly suggest that the protozoan SMT has an active site topography combining properties of the SMT1 from plants and yeast (37-47% identity). The enzymatic activation of this and other SMTs reveals that the catalytic requirements for the C-methyl reaction are remarkably versatile, whereas the inhibition studies provide a powerful approach to rational design of new anti-sleeping sickness chemotherapeutic drugs.     

Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.     

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.     

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).     

The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2;1

The Arabidopsis genome contains three distinct genes encoding sterol-C24-methyltransferases (SMTs) involved in sterol biosynthesis. The expression of one of them, STEROL METHYLTRANSFERASE 2;1, was modulated in 35S:SMT2;1 Arabidopsis in order to study its physiological function. Plants overexpressing the transgene accumulate sitosterol, a 24-ethylsterol which is thought to be the typical plant membrane reinforcer, at the expense of campesterol. These plants displayed a reduced stature and growth that could be restored by brassinosteroid treatment. Plants showing co-suppression of SMT2;1 were characterized by a predominant 24-methylsterol biosynthetic pathway leading to a high campesterol content and a depletion in sitosterol. Pleiotropic effects on development such as reduced growth, increased branching, and low fertility of high-campesterol plants were not modified by exogenous brassinosteroids, indicating specific sterol requirements to promote normal development. Thus SMT2;1 has a crucial role in balancing the ratio of campesterol to sitosterol in order to fit both growth requirements and membrane integrity.     

Diversification of sterol methyltransferase enzymes in plants and a role for β‐sitosterol in oriented cell plate formation and polarized growth

Phytosterols are classified into C24‐ethylsterols and C24‐methylsterols according to the different C24‐alkylation levels conferred by two types of sterol methyltransferases (SMTs). The first type of SMT (SMT1) is widely conserved, whereas the second type (SMT2) has diverged in charophytes and land plants. The Arabidopsis smt2 smt3 mutant is defective in the SMT2 step, leading to deficiency in C24‐ethylsterols while the C24‐methylsterol pathway is unchanged. smt2 smt3 plants exhibit severe dwarfism and abnormal development throughout their life cycle, with irregular cell division followed by collapsed cell files. Preprophase bands are occasionally formed in perpendicular directions in adjacent cells, and abnormal phragmoplasts with mislocalized KNOLLE syntaxin and tubulin are observed. Defects in auxin‐dependent processes are exemplified by mislocalizations of the PIN2 auxin efflux carrier due to disrupted cell division and failure to distribute PIN2 asymmetrically after cytokinesis. Although endocytosis of PIN2–GFP from the plasma membrane (PM) is apparently unaffected in smt2 smt3, strong inhibition of the endocytic recycling is associated with a remarkable reduction in the level of PIN2–GFP on the PM. Aberrant localization of the cytoplasmic linker associated protein (CLASP) and microtubules is implicated in the disrupted endocytic recycling in smt2 smt3. Exogenous C24‐ethylsterols partially recover lateral root development and auxin distribution in smt2 smt3 roots. These results indicate that C24‐ethylsterols play a crucial role in division plane determination, directional auxin transport, and polar growth. It is proposed that the divergence of SMT2 genes together with the ability to produce C24‐ethylsterols were critical events to achieve polarized growth in the plant lineage.     

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.     

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.     

Control of fungal sterol C-24 transalkylation: importance to developmental regulation

When cultures of Gibberella fujikuroi are incubated with 24-epiiminolanosterol the introduction of a methyl group into sterol side chains at C-24 is blocked inducing a mycelial accumulation of lanosterol and 24-desalkylsterols, i.e., having the cholestane side chain. The altered sterol composition lead to aberrant mycelial membranes resulting in growth inhibition. A compensatory physiological response to the ensuing hyphal death was induction of asexual sporulation. The results are interpreted to imply that regulation of sterol C-24 transalkylation may be a mechanism to mediate life cycle events of fungi.