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).

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).

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

, 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.

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 3β-hydroxyhopene and 3β-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.

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.

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 CH 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.

Strictly Conserved Residues in Euphorbia tirucalli β-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 β-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.

Euphorbia tirucalli β-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 β-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 β-amyrin as the dominant product.

β-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 β-amyrin synthase, which is ubiquitously found in plants, has lagged in comparison.

Correction: β-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 'β-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.

β-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 β-amyrin synthase and its catalytic mechanism remain unclear. Herein, we examined the functions of the highly conserved Phe413, Tyr259, and Trp257 residues in the β-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.

β-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 β-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 β-amyrin synthase. The bulkier ethyl group was accepted as a substrate, leading to formation of the β-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 β-amyrin skeleton 45.

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.

β-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 β-amyrin synthase were conducted to examine the role of the branched methyl groups of compound 1 in the β-amyrin biosynthesis. Substrate 8 almost exclusively afforded 30-nor-β-amyrin (>95.

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.

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.

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.

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.

β-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 β-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: (17β-H, 20S)-20-ethyl-dammara-12,24-diene 9, β-amyrin homologue 10, and the 6,6,6,6-fused tetracycle 11. The folding conformations leading to these products are discussed.

β-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.

β-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on β-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 β-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases.

Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis.

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure.

Cyclization of squalene from both termini: identification of an onoceroid synthase and enzymatic synthesis of ambrein.

The onoceroids are triterpenoids biosynthesized from squalene or (3S)-2,3-oxidosqualene by cyclization from both termini. We recently revealed that tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-β-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. Here, we reveal that BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase.

Effect of cation-π interactions and steric bulk on the catalytic action of oxidosqualene cyclase: a case study of Phe728 of β-amyrin synthase from Euphorbia tirucalli L.

The function of the active-site residues of oxidosqualene cyclases (OSCs) has been presumed mainly in light of the product distribution; however, not much research has been performed into the enzymatic activity of mutated OSCs. β-Amyrin, which is widely found in the plant kingdom, is classified as an OSC; mutational studies on β-amyrin cyclase are very limited. Six site-specific mutations targeted at the Phe728 residue of Euphorbia tirucalli β-amyrin synthase (EtAS) were constructed to inspect the function of this aromatic residue.

Identification of novel sesterterpene/triterpene synthase from Bacillus clausii.

Basic enzyme: The tetraprenyl-β-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-β-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene.

Purification, kinetics, inhibitors and CD for recombinant β-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases.

β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase.