Pathways of Phytosterol Biosynthesis

The pathway to isoprenoids (five carbon units similar to isoprene) and phytosterols in plants begins with CO₂ fixation and sugar formation (Fig. 9.3). Sugar can be converted to acetate which can be converted to isoprenoids and phytosterols in the cytosol. In producing phytosterols, acetate is converted to mevalonic acid (MVA). The MVA is phosphorylated and the carboxyl carbon is lost as CO₂ to produce Δ³-isopentyl diphosphate (IPP). However, the sugar can be converted to isoprenoids in the plastids without necessarily involving the intermediacy of acetate via the mevalonate-independent pathway [=1-deoxy xylulose-5- phosphate (DXP) pathway]. The DXP synthase is considered rate-limiting in this pathway. Carbons from Δ³-IPP can flow to the sterol or fatty acid pathways (via the MVA shunt) (Nes and Bach, 1985). HMGCoA-reductase (HMGR) is considered to be the rate-limiting enzyme of the acetate–mevalonate pathway to isoprenoids. Six of the isoprenoid units are joined to produce squalene. The C-30 olefin is converted to squalene oxide which is cyclized to 24-desalkyl sterols. The extra ‘‘methyl or ethyl’’ group at C-24 in the side-chain of phytosterols is added after formation of the first tetracycle by the action of SMT. In the phytosterol pathway, SMT is considered to be a rate-limiting enzyme.

FIGURE 9.3 Stages in the isoprenoid–phytosterol pathway and compartmentation of acetate–mevalonate pathways.

Biosynthetic tracer studies have been carried out on almost all classes of phytosterols, and in many instances cell-free preparations capable of converting Δ²⁴-sterols to methylated products are available. In almost all instances, the C-methylation processes are consistent with an ionic mechanism hypothesized by Castle et al. (1963), attesting to a common set of reactions that evolved for this class of enzyme. Detailed information about isoprenoid-sterol biosynthesis and the pathways involved in sterol side-chain construction has been obtained recently using ¹³C and ²H-labeled compounds (Kresge et al., 2005; Seo et al., 1988, 1990). Isotopic labeling experiments with stable isotopes are often used to avoid radioactive measurements and laborious chemical degradation of the sterol side-chain (Nes and Le, 1990). The sites of labeling in the end product are evident through peak enhancements in the ¹³C NMR spectra of the ¹³C-labeled sterols and reveal the route of carbon flux from the isoprenoid pathway to phytosterols.

Plant biochemists have shown that two distinct pathways to sterols exist in the cell and they are compartmentalized so that the acetate–mevalonate pathway is cytosolic and the mevalonate-independent pathway is plastidial (Arigoni et al., 1997; De-Eknamkul and Potduang, 2003; Laule et al., 2003; Lichtenthaler et al., 1997; Nes et al., 1992; Umlauf et al., 2004; Zhou and Nes, 2000). Some typical experiments to determine the pathway of phytosterol synthesis involve treating plants with [1-¹³C]glucose. The sugar is converted to [3-¹³C]pyruvate which is converted to [2-¹³C]acetate and the C-2 unit is converted in several reactions to [2,4,5-¹³C₃]Δ³ IPP; or the [3-¹³C]pyruvate is converted to [3-¹³C]glyceraldehyde 3-phosphate and this intermediate is transformed to [1,5-¹³C₂]Δ³-IPP. The labeling pattern of, for example, ergosterol derived from administering cells [1-¹³C]glucose has been found to be different in different organisms whether the Δ³-IPP is formed from the glucose breakdown product of [2-¹³C]acetate or originates directly from the intermediate DXP (Zhou and Nes, 2000; Lichtenthaler et al., 1997). When the plastid-derived intermediates are labeled with [1-¹³C]glucose, the labeling pattern of the

FIGURE 9.4 Determination of labeling patterns in isopentyl diphosphate and sterols synthesized by different pathways.

biosynthetically formed sterol molecule is predicted to be [2,6,11,12,16,18,19,23,27-¹³C₉]ergosterol. A spectrum of ergosterol, biosynthesized from [1-¹³C]glucose using the alga Prototheca wickerhamii shows enhanced peaks corresponding to nine carbon atoms (earlier we incorrectly identified the enhanced peak corresponding to C-11 and C-21 to be from [1-¹³C]glucose (Zhou and Nes, 2000); actually the enhanced peak is due to C-11 only; Fig. 9.5; spectrum c), suggesting the alga operates the mevalonate-independent pathway to sterols. Alternatively, the labeling pattern of the products of the acetate-mevalonate pathway is predicted to be [1,3,5,7,9,13,15,17,18,19,21,22,24,26,27-¹³C₁₅]sitosterol (De- Eknamkul and Potduang, 2003;Umlauf et al., 2004). Since neither C-28 nor C-29 will be labeled by these intermediates, the labeling patterns of ergosterol and sitosterol assayed with [1-¹³C]glucose will be the same (Fig. 9.4). Evidence to confirm the traditional isoprenoid–sterol pathway was obtained by administering [2-¹³C] MVA or [5-¹³C]MVAto culturedsunflower cells andnotingthenumber andpositionof the enhanced peaks in the ¹³CNMR spectrum(Fig. 9.5; spectra a and b) (Nes et al., 1992).

FIGURE 9.5 Stereochemistry of phytosterols at C-25 after 13C labeling of the ProE C-26 of Δ24-sterols.

As shown in Fig. 9.5, C-26 and C-27 of the sterol side-chain are chemically equivalent yet they are biosynthetically distinct. Seo et al. (1990) have shown that C-26 is derived from C-6 of MVA and C-27 is derived from C-2 of MVA. Appropriate rotations in the structures of these compounds generate a view of the sterol side-chain in equivalent conformation so that the stereochemistry at C-25 can be rationalized and the existence of stereodifferentiated enzymes determined from tracer studies with ¹³C-labeled substrates. Nes et al. (1992) examined the incorporation of [2-¹³C]MVA to sitosterol and found that C-26 is labeled and Seo et al. labeled C-26 in ergosterol with [2-¹³CH₃]acetate (Zhou and Nes, 2000). To confirm that the stereochemistry at C-25 is the same in sitosterol and ergosterol, acceptors [27-¹³C]zymosterol, [27-¹³C]lanosterol, and [27-¹³C]cycloartenol were prepared and assayed with cell-free preparations from plants, fungi, and algae (Guo et al., 1996; Mangla and Nes, 2000; Nes et al., 1998b; Zhou et al., 1996). The enzymatically formed side-chains of fecosterol, 24(28)-methylene lanosterol, and cyclolaudenol contained the C-25R-configuration as determined by 13C NMR spectroscopic analysis, thereby showing the ProZ C-27 of the Δ²⁴-intermediate generates the R-C-27 methyl group of the phytosterol.

Incubation of [1-¹³C]glucose with cultured cells or intact plants can generate different ¹³C-labeled species of phytosterol and the purity of the labeling pattern of the sterol can indicate the degree to which the acetate–mevalonate pathway or mevalonate-independent pathway operates and the extent of cross talk between the pathways (Arigoni et al., 1997; Laule et al., 2003). These studies indicate that the acetate–mevalonate pathway (=isoprenoid pathway) is preferred in vascular plants. This observation is further substantiated by physiology experiments with inhibitors of HMGR such as mevinolin added to radish seedlings (Bach and Lichtenthaler, 1983) or fosmidomycin, an inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, added to tobacco cells (Sauvaire et al., 1997), and by flux studies using tracer amounts of radiolabeled acetate and high concentrations of mevalonate to inhibit sterol synthesis in sorghum seedlings (Hemmerlin et al., 2003).

FIGURE 9.6 Hypothetical pathways to Δ5-phytosterols: C1 and C2 refer to the CH3 and C2H5 groups attached to C-24 of the sterol side-chain. Circled C-4 is to emphasize C-4 methyl group removal; 4,4-dimethyl sterol to 4-monomethyl sterol to 4-desmethyl sterol.

In the past several years, there have been several remarkable advances in the study of phytosterol enzymes to support the hypothesis that multiple phytosterol pathways exist in nature. The start and direction of the pathway is established by whether squalene oxide cyclizes to cycloartenol (plants and algae) or lanosterol (animals and fungi) (Fig. 9.6; Nes and McKean, 1977). Thereafter, the C-24 methylation pathways provide direction to phytosterol synthesis; ‘‘primitive’’ plants catalyze the Δ²⁵⁽²⁷⁾-route whereas advanced plants and fungi catalyze the Δ²⁴⁽²⁸⁾-route (Goad et al., 1974; Nes et al., 1977). Mass spectroscopy (MS) can be used as a first screen to determine whether ergosterol is formed by either a fungal or algal route. For example, administering [²H₃]methionine to a yeast sterol auxotroph GL7 cultured on lanosterol led to the biosynthesis of [28-²H]ergosterol (Zhou et al., 1996). The mass spectrum of the deuterated ergosterol was found to be two mass units higher than the control species, consistent with methylation at C-24 proceeding by a Δ²⁴⁽²⁸⁾-methylene intermediate. The site of introduction of methyl in the sterol side-chain was determined by inspection of the ¹H NMR spectra of ergosterol

FIGURE 9.7 Spectroscopic and chromatographic analysis of sterols.

and the corresponding deuterium-labeled ergosterol (Fig. 9.7, Panels B and A). The only signal in the ¹H NMR spectrum affected by the incorporation of deuterium in the molecule corresponds to C-28 which is lowered compared to the control. In related experiments with a cell-free preparation of SMT from yeast assayed with [²H₃-methyl]AdoMet and zymosterol, the enzymatic product [28-²H]fecosterol contained two extra deuterium atoms as determined by MS; in the ¹H NMR spectrum, the olefin peak at ca. 4.65 ppm (not shown) corresponding to C-28 was missing but it was present in the spectrum of unlabeled fecosterol (Fig. 9.7; Nes et al., 1998b). These findings show methyl from AdoMet is added to C-24 of the sterol side-chain and becomes C-28 of yeast ergosterol via a pathway that involves methylation of zymosterol to produce the Δ²⁴⁽²⁸⁾-olefin fecosterol, as expected (Goad et al., 1974). In the pathway to algal ergosterol, activity assay with [²H₃-methyl]AdoMet and cycloartenol produces an intermediate molecule cyclolaudenol that mass spectroscopy reveals contains three rather than two deuterium atoms (Zhou et al., 1996). Thus, the methylation route to fungal ergosterol proceeds by a Δ²⁴⁽²⁸⁾-route whereas the algal route proceeds by the D25(27)-route.

Phytosterols will incorporate either four (α-configuration) or five (β-configuration) deuterium atoms in the 24-ethyl group after treatment with [²H₃-methionine] (Goad et al., 1974), but in these instances the sterols can be derived from the same SMT by a similar C-24-methylation mechanism (Kaneshiro et al., 2002; Nes et al., 2003). To address whether the mechanism of sterol C-methylation leading to vascular plant campesterol (24α-methyl group) is the same as the one leading to fungal ergosterol (24β-methyl group), Nes and coworkers recovered 27-¹³C-24(28)- methylene lanosterol from activity assay of 27-¹³C-lanosterol prepared in a cellfree corn system (Guo et al., 1996). The ¹³C-labeled compound was administered to a yeast sterol auxotroph GL7 which converted the dietary supplement to [27-¹³C] ergosterol (Zhou et al., 1996). The ¹³C NMR spectrum of either the [27-¹³C]ergosterol derived by the plant- or fungal-generated [27-¹³C]-labeled intermediates were identical, indicating the phytosterol C-methylation pathway in the two organisms is similar and involves a 1,2-hydride shift from the Re-face of the original substrate undergoing methylation. Using cloned SMTs from a mutant yeast (Y81W) and wild-type soybean and Arabidopsis, a single enzyme was found to catalyze both the first and second C₁-transfer activities, and in the case of the second C₁-transfer activity the stereochemistry of the 24-ethyl group (β) in the product was shown to be opposite to that of the 24-ethyl group in sitosterol (α) (Fig. 9.6). Inevitably, the conclusion that different phytosterol pathways are present in plants and fungi stems from the fact that different enzymes control the stereochemistry of the methylated products and that the substrate affinities and catalytic competence of SMTs can be different in different organisms.