Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway

Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [(3)H]Phe was fed, the majority of metabolized [(3)H]Phe was incorporated into p-[(3)H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [(3)H]Phe and [(14)C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [(3)H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants.     

Insights into phycoerythrobilin biosynthesis point toward metabolic channeling

Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IXalpha, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme.bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.     

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis

Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope- and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.     

Genomic evidences for the existence of a phenylpropanoid metabolic pathway in Aspergillus oryzae

Plants interact with their environment by producing a diverse array of secondary metabolites. A majority of these compounds are phenylpropanoids and flavonoids which are valued for their medicinal and agricultural properties. The phenylpropanoid biosynthesis pathway proceeds with the basic C6-C3 carbon skeleton of phenylalanine, and involves a wide range of enzymes viz., phenylalanine ammonia lyase, coumarate hydroxylase, coumarate ligase, chalcone synthase, chalcone reductase and chalcone isomerase. Recently, bacteria have also been shown to contain homodimeric polyketide synthases belonging to the plant chalcone synthase superfamily linking the capabilities of plants and bacteria in the biosynthesis of flavonoids. We report here the presence of genes encoding the core enzymes of the phenylpropanoid pathway in an industrially useful fungus, Aspergillus oryzae. Although the assignment of enzyme function must be confirmed by further biochemical evidences, this work has allowed us to anticipate the phenylpropanoid metabolism profile in a filamentous fungus for the first time and paves way for research on identifying novel fungal flavonoid-like metabolites.     

Ubiquitin-independent entry into the yeast recycling pathway

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway links to vacuolar degradation of the receptor, while a ligand-dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand-dependent endocytosis mechanism. The doa4Δ mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand-dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand-dependent uptake was further indicated by analysis of receptor mutants having Lys-to-Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand-induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4 -dependent turnover of the receptor (for receptor internalized via the ligand-dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys-to-Arg mutations was noted. Following a prolonged exposure of the cells to the a-factor ligand, rather than being delivered to the vacuolar lumen, the Lys-to-Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a-factor-dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.     

Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway

The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.     

The entry point of the K-proton-transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump. The enzyme has two proton input pathways, leading from the solution on the N-side to the binuclear center. One of these pathways, the K-pathway, is used for proton uptake upon reduction of the binuclear center. It is also important for local charge compensation during reaction of the fully reduced enzyme with O2. Two different locations have been proposed to constitute the entry point of the K-pathway: near S(I-299) or near E(II-101), respectively, in the Rhodobacter sphaeroides enzyme. The experiments discussed in this study are aimed at identifying the location of the entry point. The kinetics and extent of flash-induced proton release coupled to oxidation of heme a3 (tau congruent with 2 ms at pH 8.8 in the wild-type enzyme) in the absence of O2 were investigated in the ED(II-101), SD(I-299), and KM(I-362) mutant enzymes, i.e., at the two proposed entry points and in the middle of the pathway, respectively. This reaction was completely blocked in KM(I-362), while it was slowed by factors of 25 and 40 in the ED(II-101) and SD(I-299) mutant enzymes, respectively. During reaction of the fully reduced enzyme with O2, electron transfer from heme a to the catalytic site (during P(R)-formation) was blocked in the KM(I-362) and SD(I-299)/SG(I-299) but not in the ED(II-101)/ EA(II-101) mutant enzymes. The results are interpreted as follows: Residue K(I-362) is involved in both proton transfer and charge compensation (in different reaction steps). The impaired proton release in the S(I-299) mutant enzymes is an indirect effect due to an altered environment of K(I-362). E(II-101), on the other hand, is likely to be part of the K-pathway since mutation of this residue results in impaired proton release but does not affect the P(R) formation kinetics; i.e., the properties of K(I-362) are not altered. Consequently, we conclude that the entry point of the K-pathway is located near E(II-101).     

Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway

The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4′-O-β-d-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104–240 µg/g fresh weight in SM (F₂). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.     

Depalmitoylation of Ykt6 prevents its entry into the multivesicular body pathway

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo . The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and – in contrast to the wild-type protein – does not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.     

Energy dissipation and radical scavenging by the plant phenylpropanoid pathway

Environmental stresses such as high light, low temperatures, pathogen infection and nutrient deficiency can lead to increased production of free radicals and other oxidative species in plants. A growing body of evidence suggests that plants respond to these biotic and abiotic stress factors by increasing their capacity to scavenge reactive oxygen species. Efforts to understand this acclimatory process have focused on the components of the 'classical' antioxidant system, i.e. superoxide dismutase, ascorbate peroxidase, catalase, monodehydroascorbate reductase, glutathione reductase and the low molecular weight antioxidants ascorbate and glutathione. However, relatively few studies have explored the role of secondary metabolic pathways in plant response to oxidative stress. A case in point is the phenylpropanoid pathway which is responsible for the synthesis of a diverse array of phenolic metabolites such as flavonoids, tannins, hydroxycinnamate esters and the structural polymer lignin. These compounds are often induced by stress and serve specific roles in plant protection, i.e. pathogen defence, ultraviolet screening, antiherbivory, or structural components of the cell wall. This review will highlight a novel antioxidant function for the taxonomically widespread phenylpropanoid metabolite chlorogenic acid (CGA; 5-O-caffeoylquinic acid) and assess its possible role in abiotic stress tolerance. The relationship between CGA biosynthesis and photosynthetic carbon metabolism will also be discussed. Based on the properties of this model phenolic metabolite, we propose that under stress conditions phenylpropanoid biosynthesis may represent an alternative pathway for photochemical energy dissipation that has the added benefit of enhancing the antioxidant capacity of the cell.     

A 'distributed degron' allows regulated entry into the ER degradation pathway.

Protein degradation is employed in both regulation and quality control. Regulated degradation of specific proteins is often mediated by discrete regions of primary sequence known as degrons, whereas protein quality control involves recognition of structural features common to damaged or misfolded proteins, rather than specific features of an individual protein. The yeast HMG-CoA reductase isozyme Hmg2p undergoes stringently regulated degradation by machinery that is also required for ER quality control. The 523 residue N-terminal transmembrane domain of Hmg2p is necessary and sufficient for regulated degradation. To understand how Hmg2p undergoes regulated degradation by the ER quality control pathway, we analyzed over 300 mutants of Hmg2p. Regulated degradation of Hmg2p requires information distributed over the entire transmembrane domain. Accordingly, we refer to this determinant as a 'distributed' degron, which has functional aspects consistent with both regulation and quality control. The Hmg2p degron functions in the specific, regulated degradation of Hmg2p and can impart regulated degradation to fusion proteins. However, its recognition is based on dispersed structural features rather than primary sequence motifs. This mode of targeting has important consequences both for the prediction of degradation substrates and as a potential therapeutic strategy for targeted protein degradation using endogenous degradation pathways.     

Characterization of glycolysis and pentose phosphate pathway activity during sperm entry into the mouse oocyte

Studying the events that occur during gamete fusion and sperm decondensation in the oocyte remains difficult because sperm-oocyte fusion and subsequent sperm decondensation represent a short part of the fertilization process, and their exact timing is difficult to determine. There is therefore a need for greater understanding of the events that occur during this period. The main purpose of this study was to examine the metabolic aspects of this time frame by characterizing glucose metabolism (glycolytic and pentose phosphate pathway [PPP] activities) during sperm fusion and decondensation into zona-free oocytes in mice. The metabolism of glucose through both glycolysis and the PPP was measured in ovulated MII oocytes, free of cumulus cells, and the levels of glucose metabolized were found to be low. Upon sperm entry, both glycolytic and PPP activity increased substantially. To determine whether this elevation in glucose metabolism was part of the activation process, the metabolism of parthenogenetically activated oocytes was measured, and no increase in metabolism was observed. The characterization of glucose metabolism during sperm fusion and decondensation into the oocyte, and comparison to parthenogenetically activated oocytes, showed that the fertilizing sperm is responsible for an increase in both glycolytic and PPP activity during fusion and/or decondensation. The significance of this observation during the fertilization process and for the developing embryo is as yet unclear and warrants further investigation.     

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.     

Metabolon formation and metabolic channeling in the biosynthesis of plant natural products

Metabolon formation and metabolic channeling in plant secondary metabolism enable plants to effectively synthesize specific natural products and to avoid metabolic interference. Channeling can involve different cell types, take advantage of compartmentalization within the same cell or proceed directly within a metabolon. New experimental approaches document the importance of channeling in the synthesis of isoprenoids, alkaloids, phenylpropanoids, flavonoids and cyanogenic glucosides. Metabolon formation and metabolic channeling in natural-product synthesis facilitate attempts to genetically engineer new pathways into plants to improve their content of valuable natural products. They also offer the opportunity to introduce new traits by genetic engineering to produce plant cultivars that adhere to the principle of substantial equivalence.     

Active site mapping and substrate channeling in the sterol methyltransferase pathway

Sterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified from Escherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor [3-3H]26,27-dehydrozymosterol (26,27-DHZ). A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18 reverse phase high performance liquid chromatography. Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling. Equilibration studies indicated that zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereas S-adenosylmethionine was bound only to the native SMT, K(d) of about 2 microm. Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26')-trienol as well as 26-homocholesta-8(9),26(26')-3beta,24beta-dienol, and in the case of D79L and E82L mutants, zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol. The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of (1)H and (13)C NMR techniques. A K(m) of 15 microm, K(cat) of 8 x 10(-4) s(-1), and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents. Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the zymosterol high energy intermediate as a consequence of less than perfect control. Alternatively, the gain in enzyme function resulting from the production of a delta(25(27))-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.