Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC _Aac ), Acetobacter pasteurianus (SHC _Apa ), Streptomyces coelicolor (SHC _Sco ) and Bradyrhizobium japonicum (SHC _Bja ) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.     

Non-specific biosynthesis of gammacerane derivatives by a cell-free system from the protozoon Tetrahymena pyriformis. Conformations of squalene, (3S)-squalene epoxide and (3R)-squalene epoxide during the cyclization

1. A cell-free system from the protozoon Tetrahymena pyriformis was incubated with either [12-3H]squalene or (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene. Squalene was cyclized into tetrahymanol whereas racemic squalene epoxide was transformed into gammacerane-3 alpha,21 alpha-diol and gammacerane-3 beta,21 alpha-diol. After cyclization of (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene, both epimeric gammaceranediols were labelled with a tritium atom located at C-3, showing that no isomerization via a 3-oxo compound occurred. 2. The proton NMR spectra of the cyclization products of synthetic (2E, 22E)-(1,1,1,24,24,24-2H6)squalene and (RS)-(22E)-2,3-epoxy-2,3-dihydro-(1,1,1,24,24,24-2H6)squalene show that squalene and the (3S)enantiomer of its epoxide are cyclized in an all pre-chair conformation, whereas the (3R) enantiomer of squalene epoxide is cyclized in a pre-boat conformation as concerns the cycle A. 3. The squalene cyclase of T. pyriformis presents the same lack of substrate specificity as the cyclase of Acetobacter pasteurianum: in addition to squalene, its normal substrate, it also cyclizes both enantiomers of its epoxide. This conformational versatility is characteristic of squalene cyclases but no longer exists in the squalene epoxide cyclases from eukaryotes.     

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).     

Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti

The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.     

Point mutation of (+)-germacrene A synthase from <Emphasis Type="Italic">Ixeris dentata</Emphasis>

Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the ₄₃₃TSA₄₃₅ triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity.     

Enzymatic (13)C labeling and multidimensional NMR analysis of miltiradiene synthesized by bifunctional diterpene cyclase in Selaginella moellendorffii

Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-(13)C(6)]mevalonate, all carbons were labeled with (13)C stable isotope (>99%). The fully (13)C-labeled product was subjected to (13)C-(13)C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and multidimensional (13)C NMR analyses of completely (13)C-labeled compound are powerful methods for biosynthetic studies.     

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species ( Stigmatella , Gemmata and Methylococcus ) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.     

Biosynthesis of monoterpenes. Stereochemical implications of acyclic and monocyclic olefin formation by (+)- and (-)-pinene cyclases from sage

(+)-Pinene cyclase from sage (Salvia officinalis) catalyzes the isomerization and cyclization of geranyl pyrophosphate to (+)-alpha-pinene and (+)-camphene, and to lesser amounts of (+)-limonene, myrcene, and terpinolene, whereas (-)-pinene cyclase from this tissue catalyzes the conversion of the acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene, and to lesser quantities of (-)-limonene, myrcene, and terpinolene. The bicyclic products of these enzymes (pinene and camphene) are derived via the cyclization of the cisoid, anti-endo-conformers of the bound, tertiary allylic intermediates (3R)-linalyl pyrophosphate [+)-pinene cyclase) and (3S)-linalyl pyrophosphate [-)-pinene cyclase). When challenged with either enantiomer of linalyl pyrophosphate or with neryl pyrophosphate (cis-isomer of geranyl pyrophosphate) as substrate, both pinene cyclases synthesize disproportionately high levels of acyclic olefins (myrcene and ocimene) and monocyclic olefins (limonene and terpinolene), compared with the product mixtures generated from the natural geranyl precursor. Resolution of the limonene derived from linalyl pyrophosphate and neryl pyrophosphate demonstrated that this monocyclic olefin was formed via conformational foldings in addition to the cisoid,anti-endo-pattern. These results indicate that the alternate substrates are ionized by the cyclases prior to their achieving the optimum orientation for bicyclization. In the case of geranyl pyrophosphate, a preassociation mechanism is suggested in which optimum folding of the terpenyl chain precedes the initial ionization step.     

Diterpene cyclases and the nature of the isoprene fold

The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site‐directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ～20 α‐helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ‐domain structure together with a third, α‐domain, forming an αβγ structure, and in H⁺‐initiated cyclases, there is an EDxxD‐like Mg²⁺/diphosphate binding motif located in the γ‐domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.     

Identification and characterization of the pseudopterosin diterpene cyclase, elisabethatriene synthase, from the marine gorgonian, Pseudopterogorgia elisabethae

The pseudopterosins are diterpene glycosides isolated from the marine gorgonian, Pseudopterogorgia elisabethae, which exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. Previously, we isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. Identification of this metabolite, and the conversion of labeled geranylgeranyl diphosphate to elisabethatriene, provided us with an assay to guide the isolation of the enzyme responsible for this cyclization. The soluble protein preparation from P. elisabethae has been partially purified (approximately 15,000-fold) using a combination of low-resolution anion-exchange, low-resolution hydrophobic interaction, high-resolution hydroxyapatite, and high-resolution anion-exchange chromatography. The diterpene cyclase was identified by comparing the molecular weight from gel permeation chromatography (approximately 47,000Da) with those of protein bands from purified fractions using SDS-PAGE gel electrophoresis. Kinetic analysis and evaluation of amino acid inhibition studies indicated that the enzyme displays similar characteristics to other terpenoid cyclases isolated from terrestrial sources. This report represents the first purification and characterization of a terpene biosynthetic enzyme from a marine invertebrate.     

Squalene-hopene cyclases

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.     

The triterpene cyclase protein family: a systematic analysis

Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction.     

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.     

Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95 ? resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally occurring, noncanonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C(11) homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and endo-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries.     

Recent advances regarding diterpene cyclase genes in higher plants and fungi

Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H(+)-ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.     

Characterization of the adenylation site in the RNA 3'-terminal phosphate cyclase from Escherichia coli

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.     

Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged. The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide. Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction. These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7). As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway. Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase: a single-point mutation converts aristolochene synthase into an (E)-beta-farnesene synthase

Sesquiterpene cyclases, many of which share significant structural similarity, catalyze the cyclization reactions of the universal alicyclic precursor farnesyl pyrophosphate to produce more than 300 different hydrocarbon skeletons with high regio- and stereospecificity. The molecular basis of this exquisite specificity is not well-understood, but the conformation adopted by FPP in the active site of a sesquiterpene cyclase is thought to be an important determinant of the reaction pathway. Aristolochene synthase (AS) from Penicillium roqueforti catalyzes the cyclization of farnesyl pyrophosphate to the bicyclic sesquiterpene aristolochene. The X-ray structure of AS suggested that the steric bulk of residue 92 was central in binding of FPP to the active site of AS in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused Decalin S-germacrene A. We demonstrate here that reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depended linearly on the size of the residues at position 92. ASY92A, in which Tyr92 had been replaced with Ala, produced almost 80% of alicyclic sesquiterpenes, suggesting an energetic separation of less than 0.8 kcal/mol between the cyclic and noncyclic reaction pathways. A mechanism by which FPP binds to the mutant enzymes in an extended conformation is proposed to explain the altered selectivity. The mutants also produced small amounts of additional hydrocarbons with a molecular weight of 204, namely, alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene. The production of (E)-beta-farnesene and beta-bisabolene suggested that the initial cyclization of FPP to germacrene A in AS proceeded in a stepwise fashion through farnesyl cation.