Enzymatic syntheses of novel carbocyclic scaffolds with a 6,5 + 5,5 ring system by squalene-hopene cyclase.

Squalene-hopene cyclase (SHC) converts acyclic squalene 1 into the 6,6,6,6,5-fused pentacyclic triterpenes hopene and hopanol. Previously, we reported the polycyclization products 14-17 of 27-norsqualene (13a) and 28-norsqualene (13b) by SHC, and suggested the importance of Me-27 of 1 for the normal polycyclization pathway. To further ensure the theory, (3R,S)-27-noroxidosqualenes (18 and 19) were incubated, and the structures of products 20-25 thus obtained prompted us to reinvestigate the SHC reaction of 13a (13b). One new product 29, composed of a 6,5 + 5,5 ring system with 13α-H and 17α-H, was obtained from 13a in addition to both the previously isolated products 14-17 and the 6,6,6,5-fused tetracyclic dammarenyl compounds, which were overlooked before. We propose the name "nor-allodammarane" for this novel tetracyclic 6,5 + 5,5 ring system and the name "nor-allogammacerane" for the pentacyclic 6,5 + 5,5 + 6 ring system. The stereochemistry of 29 indicated that 13a folded in the following chair-boat-boat-boat conformation: 10α-H, 11ß-H; 14α-H, 15ß-Me; 18α-H and 19ß-Me, which further allowed us to predict the configuration of 20R for 14 and that of 20S for 15. Substrates 18 and 19 were also cyclized only into allodammarane scaffolds 20-25, and all the structures of 20-25 further indicated that 18 and 19 also folded in the same conformation as 13a, providing further evidence that Me-27 groups of 1 and oxidosqualene are essential for the normal polycyclization pathway by SHC.

Oryza sativa Parkeol Cyclase: Changes in the Substrate-Folding Conformation and the Deprotonation Sites on Mutation at Tyr257: Importance of the Hydroxy Group and Steric Bulk.

Y257 of Oryza sativa parkeol synthase (OsOSC2) corresponds to H234 of Saccharomyces cerevisiae lanosterol cyclase (ScLAS), which is believed to be responsible for the final deprotonation reaction. An Ala mutant afforded nine tetracyclic skeletons as the main products; they consisted of protostadienol scaffolds with both 17R and 17S configurations and both 20R and 20S configurations, as well as a pair of 20R- and 20S-configured parkeols. The production of 20R- and 20S-configured tetracycles (59:40 ratio) through the action of the Y257A mutant indicated that the substrate folding had been altered from a chair-boat-chair-chair (a normal folding pattern) to a chair-boat-chair-boat structure (an unusual folding pattern). Other mutants with different steric bulks also yielded both 20R- and 20S-configured tetracycles. Thus, the primary function of Y257 appears to be to impose a normal chair structure at the D-ring site through having appropriate steric bulk. In contrast, mutations at H234 of ScLAS were reported to cause no conformational changes. The OsOSC2 Phe mutant also yielded 20R- and 20S-configured parkeols (25:33 ratio), thus suggesting that the OH group of Y257 can form hydrogen bonds with other amino acids to force a chair conformation at the D-ring site, and this variant also gave 20R- and 20S-configured parkeols in a high yield (60 %). Y257 is unlikely to act as a base to abstract H-11 and stabilize the transient cation through cation-π interactions. Thus, the catalytic roles of Y257 are significantly different from those of H234 of ScLAS.

Chemical structure of cichorinotoxin, a cyclic lipodepsipeptide that is produced by Pseudomonas cichorii and causes varnish spots on lettuce.

Pseudomonas cichorii, which causes varnish spots on lettuce and seriously damages lettuce production during the summer season in the highland areas of Japan (e.g., Nagano and Iwate prefectures) was isolated. The structure of a toxin produced by this organism was analyzed based on the detailed evaluation of its 2D NMR and FABMS spectra, and this compound has not been reported previously. We propose the name cichorinotoxin for this toxin. In conjunction with the D or L configurations of each amino acid, which were determined by Marfey's method, we propose the structure of cichorinotoxin to be as follows: 3-hydroxydecanoyl-(Z)-dhThr1-D-Pro2-D-Ala3-D-Ala4-D-Ala5-D-Val6-D-Ala7-(Z)-dhThr8-Ala9-Val10-D-Ile11-Ser12-Ala13-Val14-Ala15-Val16-(Z)-dhThr17-D-alloThr18-Ala19-L-Dab20-Ser21-Val22, and an ester linkage is present between D-alloThr18 and Val22 (dhThr: 2-aminobut-2-enoic acid; Dab: 2,4-diaminobutanoic acid). Thus, the toxin is a lipodepsipeptide with 22 amino acids. The mono- and tetraacetate derivatives and two alkaline hydrolysates, compounds A and B, were prepared. We discuss here the structure-activity relationships between the derivatives and their necrotic activities toward lettuce.

Mutated variants of squalene-hopene cyclase: enzymatic syntheses of triterpenes bearing oxygen-bridged monocycles and a new 6,6,6,6,6-fusded pentacyclic scaffold, named neogammacerane, from 2,3-oxidosqualene.

Squalene-hopene cyclase (SHC) catalyzes the conversion of acyclic squalene molecule into a 6,6,6,6,5-fused pentacyclic hopene and hopanol. SHC is also able to convert (3S)-2,3-oxidosqualene into 3ß-hydroxyhopene and 3ß-hydroxyhopanol and can generate 3α-hydroxyhopene and 3α-hydroxyhopanol from (3R)-2,3-oxidosqualene. Functional analyses of active site residues toward the squalene cyclization reaction have been extensively reported, but investigations of the cyclization reactions of (3R,S)-oxidosqualene by SHC have rarely been reported. The cyclization reactions of oxidosqualene with W169X, G600F/F601G and F601G/P602F were examined. The variants of the W169L generated new triterpene skeletons possessing a 7-oxabicyclo[2.2.1]heptane moiety (oxygen-bridged monocycle) with (1S,2S,4R)- and (1R,2S,4S)-stereochemistry, which were produced from (3R)- and (3S)-oxidosqualenes, respectively. The F601G/P602F double mutant also furnished a novel triterpene, named neogammacer-21(22)-en-3ß-ol, consisting of a 6,6,6,6,6-fused pentacyclic system, in which Me-29 at C-22 of the gammacerane skeleton migrated to C-21. We propose to name this novel scaffold neogammacerane. The formation mechanisms of the enzymatic products from 2,3-oxidosqualene are discussed.

Alicyclobacillus acidocaldarius Squalene-Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3-Oxidosqualene.

The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,ß-hydroxyhopenes and 3α,ß-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.

Squalene-Hopene Cyclase: Mechanistic Insights into the Polycyclization Cascades of Squalene Analogs Bearing Ethyl and Hydroxymethyl Groups at the C-2 and C-23 Positions.

Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene 1 into 6,6,6,6,5-fused pentacyclic hopene 2 and hopanol 3. To elucidate the binding sites for the terminal positions of 1, four analogs, having the larger ethyl (Et) and the hydrophilic CH2 OH groups at the 23E or 23Z positions of 1, were incubated with SHC. The analog with the Et group at the 23E position (23E-Et-1) yielded two tetra- and three pentacyclic products; however, the analog possessing the Et group at the 23Z position (23Z-Et-1) gave two hopene homologs and the neohopane skeleton, but no hopanol homologs. Hopene homolog (C31 ) was generated from 23E-Et-1 by deprotonation from 23Z-Me (normal cyclization cascade). Intriguingly, the same homolog was also generated from the geometrical isomer 23Z-Et-1, indicating C-C bond rotation about the C-21-C-22 axis of the hopanyl cation and the more compact nature of the binding domain at 23Z compared with 23E. On the other hand, analogs with the CH2 OH group gave novel hopane skeletons having 1-formylethyl and 1-hydroxyprop-2-en-2-yl residues at C-21. Products bearing an aldehyde group were generated in higher yield from 23Z-CH2 OH-1 (89 %), than from 23E-CH2 OH-1 (26 %). The significant yield (26 %) of the aldehyde products from 23E-CH2 OH-1 indicated that C-C bond rotation had occurred owing to the absence of hydrophobic interactions between the hydrophilic 23E-CH2 OH and its binding site. The polycyclization mechanisms of the four different analogs are discussed.

Strictly Conserved Residues in Euphorbia tirucalli ß-Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation-π Interaction and CH-π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture.

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli ß-amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild-type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π-electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation-π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E-ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56-78 % of wild-type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH-π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation-π interaction.

Euphorbia tirucalli ß-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH-π Interaction between Val483 and Trp534 for Catalytic Action.

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli ß-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, afforded ß-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation-π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH-π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.

ß-Amyrin biosynthesis: catalytic mechanism and substrate recognition.

The enzymatic polycyclization reactions catalyzed by oxidosqualene (OXSQ) cyclases (OSCs) proceed with complete regio- and stereospecificity, leading to the formation of new C-C bonds and chiral centers and to the generation of diverse polycyclic sterols and triterpenoids. The diverse structural array is remarkable, and approximately 150 different carbon frameworks have been found. Detailed investigations on squalene-hopene cyclase (SHC) and lanosterol synthase (LaS) have been reported, but progress in the study of ß-amyrin synthase, which is ubiquitously found in plants, has lagged in comparison. In the past several years, remarkable advances in ß-amyrin biosynthetic studies have been made. In this review, the catalytic mechanism and substrate recognition of ß-amyrin synthase, as revealed by site-directed mutagenesis and substrate analog experiments, are outlined and compared with those of LaS and SHC to highlight the features of ß-amyrin synthase.

Correction: ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

Correction for 'ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade' by Ryousuke Ito et al., Org. Biomol. Chem., 2017, 15, 177-188.

ß-Amyrin synthase from Euphorbia tirucalli L. functional analyses of the highly conserved aromatic residues Phe413, Tyr259 and Trp257 disclose the importance of the appropriate steric bulk, and cation-π and CH-π interactions for the efficient catalytic action of the polyolefin cyclization cascade.

Many of the functions of the active site residues in ß-amyrin synthase and its catalytic mechanism remain unclear. Herein, we examined the functions of the highly conserved Phe413, Tyr259, and Trp257 residues in the ß-amyrin synthase of Euphorbia tirucalli. The site-specific mutants F413V and F413M [corrected] showed nearly the same enzymatic activities as the wild type, indicating that π-electrons are not needed for the catalytic reaction. However, the F413A [corrected] mutant yielded a large amount of the tetracyclic dammarane skeleton, with decreased production of ß-amyrin. This indicates that the Phe413 [corrected] residue is located near the D-ring formation site and works to position the oxidosqualene substrate correctly within the reaction cavity. On the other hand, the major catalysis-related function of the Tyr259 and Trp257 residues is to yield their π-electrons to the cationic intermediates. The Y259F variant showed nearly equivalent activity to that of the wild type, but aliphatic mutants such as the Ala, Val, and Leu variants showed significantly decreased the activity and yielded the tetracyclic dammarane scaffold, strongly demonstrating that the Tyr259 residue stabilizes the baccharenyl secondary cation via cation-π interaction. The aliphatic variants of Trp257 exhibited remarkably decreased enzymatic activity, and lupeol was produced in a high production ratio, indicating that Trp257 stabilizes the oleanyl cation via cation-π interaction. The aromatic Phe and Tyr mutants exhibited high activities owing to their more increased π-electron density relative to that of the aliphatic mutants, but lupeol was produced in a significantly high yield besides ß-amyrin. The Trp residue is likely to be responsible for the robust binding of Me-30 through CH-π interaction. The decreased π-electron density of the Phe and Tyr mutants compared to that of Trp would have resulted in the high production of lupeol.

ß-Amyrin Biosynthesis: Promiscuity for Steric Bulk at Position 23 in the Oxidosqualene Substrate and the Significance of Hydrophobic Interaction between the Methyl Group at Position 30 and the Binding Site.

To examine how the sterics at the 23 position of (3S)-2,3-oxidosqualene 1 influence the polycyclization cascade in ß-amyrin biosynthesis, substrate analogues substituted with an ethyl group (10, 11), a hydrogen atom (12, 13), or a propyl residue (14) at the 23 position were incubated with ß-amyrin synthase. The bulkier ethyl group was accepted as a substrate, leading to formation of the ß-amyrin skeleton (42, 43) without truncation of the multiple cyclization reactions. Analogue 13, possessing a hydrogen atom and an ethyl group at the 23E and 23Z positions, respectively, was also converted into the ß-amyrin skeleton 45. However, the analogue lacking an ethyl group at the 23Z position (12) underwent almost no conversion, strongly indicating that an alkyl group must exist at the Z position. The cyclization of the analogue with a propyl substituent at the Z position (14) was poor. Analogue 15 possessing CH2OH at the 23E position afforded a new compound 47 in a high yield as a result of trapping of the final oleanyl cation. Conversely, 16 with 23Z-CH2OH afforded novel compounds 48-50 in low yields, which resulted from the intermediary dammarenyl and baccharenyl cations. Therefore, the hydrophobic interaction between the 23Z-alkyl group and its binding site (possibly via CH/π interaction) is critical for adopting the correct chair-chair-chair-boat-boat conformation and for the full cyclization cascade.

Further Insight into Polycyclization Cascades of Acyclic Geranylfarnesol and its Acetate by Squalene-hopene Cyclase from Alicyclobacillus acidocaldarius.

The enzymatic reactions of geranylfarnesol (8) and its acetate 9, classified as sesterterpenes (C25), using squalene-hopene cyclase (SHC) were investigated. The enzymatic reaction of 8 afforded 6/6-fused bicyclic 20, 6/6/6-fused tricyclic 21, and 6/6/6/6-fused tetracyclic compounds 22 and 23 as the main products (35% yield), whereas that of 9 afforded two 6/6/6-fused tricyclic compounds 24 and 25 in a high yield (76.3%) and a small amount (5.0%) of 26 (the acetate of 22). A significantly higher conversion of 9 indicates that the arrangement of the substrate in the reaction cavity changed. The lipophilic nature and/or the bulkiness of the acetyl group may have changed its binding with SHC, thus placing the terminal double bond of 9 in the vicinity of the DXDD motif of SHC, which is responsible for the proton attack on the double bond to initiate the polycyclization reaction. The results obtained for 8 are different to some extent than those reported by Shinozaki et al. The products obtained in this study were deprotonated compounds; however, the products reported by Shinozaki et al. were hydroxylated compounds.

ß-Amyrin Biosynthesis: The Methyl-30 Group of (3S)-2,3-Oxidosqualene Is More Critical to Its Correct Folding To Generate the Pentacyclic Scaffold than the Methyl-24 Group.

Oxidosqualene cyclases catalyze the transformation of oxidosqualene (1) into numerous cyclic triterpenes. Enzymatic reactions of 24-noroxidosqualene (8) and 30-noroxidosqualene (9) with Euphorbia tirucalli ß-amyrin synthase were conducted to examine the role of the branched methyl groups of compound 1 in the ß-amyrin biosynthesis. Substrate 8 almost exclusively afforded 30-nor-ß-amyrin (>95.5 %), which was produced through a normal cyclization pathway, along with minor products (<4.5 %). However, a lack of the Me-30 group (analogue 9) resulted in significantly high production of premature cyclization products, including 6/6/6/5-fused tetracyclic and 6/6/6/6/5-fused pentacyclic skeletons (64.6 %). In addition, the fully cyclized product (35.4 %) having the 6/6/6/6/6-fused pentacycle was produced; however, the normally cyclized product, 29-nor-ß-amyrin was present in only 18.6 % of these products. The conversion yield of substrate 8 possessing a Z-Me group at the terminus was approximately twofold greater than that of compound 9 with an E-Me group. Thus, the Me-30 group is essential for the correct folding of a chair-chair-chair-boat-boat conformation of compound 1 for the production of the ß-amyrin scaffold, whereas the Me-24 group exerts little influence on the normal polycyclization cascade. Here, we show that the Me-30 group plays critical roles in constructing the ordered architecture of a chair-chair-chair-boat-boat structure, in facilitating the ring-expansion reactions, and in performing the final deprotonation reaction at the correct position.

Identification of a new diterpene biosynthetic gene cluster that produces O-methylkolavelool in Herpetosiphon aurantiacus.

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)-kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)-O-methylkolavelool from (+)-kolavelool, so this enzyme was an O-methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)-O-methylkolavelool biosynthetic gene cluster.

Non-contrast myocardial perfusion using a novel 4D magnetic resonance arterial spin labeling technique: initial experience.

The aim of this study is to develop a novel non-contrast 4-dimensional MR arterial spin labeling (4D-ASL) technique (3D acquisition and time) and to investigate myocardial perfusion on healthy volunteers without administration of contrast materials. A non-contrast 4D-ASL technique was developed using the time-spatial labeling inversion pulse (Time-SLIP) to obtain myocardium perfusion of eight volunteers at 1.5 T. The tagging slab was placed on the proximal ascending aorta to invert the blood magnetization and mid-ventricle 3D images at diastolic phase were acquired with multiple tagging delays. The time resolved 3D images with various inversion times (TI) were registered and segmented for the visualization of myocardial signal changes along the TI, and perfusion curves were generated to identify the perfusion peaks. Blood flow from basal to apical slices was observed in all volunteers. Peak flow at the mid-ventricle was observed 200-400 ms after the blood was tagged at the aortic root blood. After the perfusion peak, all signals returned to the base line. The 4D Time-SLIP technique permits non-contrast perfusion images with high temporal resolution, which may potentially differentiate normal from diseased myocardium.

Biochemical characterization of the water-soluble squalene synthase from Methylococcus capsulatus and the functional analyses of its two DXXD(E)D motifs and the highly conserved aromatic amino acid residues.

Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction.

Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum.

We report the first X-ray crystal structure of ent-kaur-16-ene synthase from Bradyrhizobium japonicum, together with the results of a site-directed mutagenesis investigation into catalytic activity. The structure is very similar to that of the α domains of modern plant terpene cyclases, a result that is of interest since it has been proposed that many plant terpene cyclases may have arisen from bacterial diterpene cyclases. The ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues. A bisphosphonate inhibitor binds to the same site. In the kaurene synthase from the moss Physcomitrella patens, 16-α-hydroxy-ent-kaurane as well as kaurene are produced since Leu and Tyr in the P. patens kaurene synthase active site are replaced by smaller residues enabling carbocation quenching by water. Overall, the results represent the first structure determination of a bacterial diterpene cyclase, providing insights into catalytic activity, as well as structural comparisons with diverse terpene synthases and cyclases which clearly separate the terpene cyclases from other terpene synthases having highly α-helical structures.

ß-Amyrin biosynthesis: the critical role of steric volume at C-19 of 2,3-oxidosqualene for its correct folding to generate the pentacyclic scaffold.

The effect of the steric volume at C-19 of (3S)-2,3-oxidosqualene 1 on the polycyclization reaction by ß-amyrin synthase was examined. The substrate analogs, in which the methyl group at C-19 of 1 was substituted by an ethyl group and hydrogen atom, were converted into the following three new compounds: (17ß-H, 20S)-20-ethyl-dammara-12,24-diene 9, ß-amyrin homologue 10, and the 6,6,6,6-fused tetracycle 11. The folding conformations leading to these products are discussed.

ß-Amyrin synthase from Euphorbia tirucalli. Steric bulk, not the π-electrons of Phe, at position 474 has a key role in affording the correct folding of the substrate to complete the normal polycyclization cascade.

ß-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on ß-amyrin synthase are very limited as compared with those of squalene-hopene cyclase and lanosterol synthase. This study was conducted to elucidate the function of the F474 residue of Euphorbia tirucalli ß-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases. Nine site-specific variants with Gly, Ala, Val, Leu, Met, Tyr, Trp, His, and Thr were constructed. We isolated 9 products from these mutants in addition to ß-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of ß-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9ßH)-polypoda-7,13,17,21-tetraen-3ß-ol and (9ßH)-polypoda-8(26),13,17,21-tetraen-3ß-ol, which are generated from a chair-boat folding conformation. This is the first report describing the conformational change from the chair-chair into the chair-boat folding conformation among the reported mutagenesis studies of oxidosqualene cyclases. Substitution with aliphatic amino acids lacking π-electrons such as Val, Leu, and Met led to a significantly decreased production of bicyclic compounds, and in turn exhibited a higher production of ß-amyrin. Furthermore, the Leu and Met variants exhibited high enzymatic activities: ca. 74% for Leu and ca. 91% for Met variants as compared to the wild-type. These facts unambiguously demonstrate that the major role of Phe474 is not to stabilize the transient cation via cation-π interaction, but is to confer the appropriate steric bulk near the B-ring formation site, leading to the completion of the normal polycyclization pathway without accumulation of abortive cyclization products.