Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071

Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds. It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation. Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs. We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis. Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane. The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling. The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.     

Origin of structural diversity in natural triterpenes: direct synthesis of seco-triterpene skeletons by oxidosqualene cyclase

At1g78500, one of the oxidosqualene cyclase (OSC) homologues from Arabidopsis thaliana, was expressed in a lanosterol synthase-deficient yeast strain and the products were analyzed. In addition to the known triterpenes, this OSC was found to produce two new triterpenes, the structures of which were determined by NMR and MS analyses. The new triterpenes are C-ring-seco-beta-amyrin (1) and C-ring-seco-alpha-amyrin (2) and named beta-seco-amyrin and alpha-seco-amyrin, respectively. beta-seco-Amyrin is produced from the oleanyl cation through bond cleavage between C8 and C14, and alpha-seco-amyrin is produced from the ursanyl cation in the same manner. Together with Grob fragmentation catalyzed by another OSC (marneral synthase) from A. thaliana, the formation of seco-amyrins by this OSC revealed that OSCs not only catalyze carbon-carbon bond formations and Wagner-Meerwein rearrangements but also cleave preformed ring systems in cationic intermediates. Based on this information, direct production of other natural seco-triterpenes by OSCs is proposed.     

Production of triterpene acids by cell suspension cultures of Olea europaea

Olive (Olea europaea) contains large quantity of triterpene acids including oleanolic acid (6) as a major one. Varieties of biological activities exhibited by triterpene acids attracted our attentions, especially from pharmaceutical viewpoints. Cell culture of olive plant was induced and its triterpene constituents were studied. From the cell suspension cultures, six ursane type triterpene acids; ursolic acid (9), pomolic acid (10), rotundic acid (11), tormentic acid (12), 2alpha-hydroxyursolic acid (13) and 19alpha-hydroxyasiatic acid (14), and two oleanane type acids; oleanolic acid and maslinic acid (7), have been isolated. Quantity of ursane type triterpene acids produced by cell cultures was larger than that of oleanane type. Further, a multifunctional oxidosqualene cyclase (OSC) named OEA was cloned by homology based PCRs from the same cultured cells. Major product of OEA is alpha-amyrin (ursane skeleton), showing good accordance to higher content of ursane-type triterpene acids in the cultured cells, and strongly suggesting OEA to be a major contributor OSC for their production.     

Novel squalene-hopene cyclase inhibitors derived from hydroxycoumarins and hydroxyacetophenones

Squalene-hopene cyclase (SHC) is a useful model enzyme for predicting molecular interactions with oxidosqualene cyclase (OSC). Structure--activity relationships were investigated for numerous coumarin-derived inhibitors of SHC, and structural simplifications are suggested. Both umbelliferone and 2,4-dihydroxyacetophenone provide convenient starting nuclei for the design of SHC inhibitors. Derivatives bearing an omega-epoxyfarnesyl moiety or just a plain alkyl chain showed an inhibitory effect on a recombinant SHC from Alicyclobacillus acidocaldarius expressed in Escherichia coli.     

Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and product distribution

BACKGROUND: Two regions of squalene-hopene cyclase (SHC) were examined to define roles for motifs posited to be responsible for initiation and termination of the enzyme-catalyzed polyolefinic cyclizations. Specifically, we first examined the triple mutant of the DDTAVV motif, a region deeply buried in the catalytic cavity and thought to be responsible for the initiation of squalene cyclization. Next, four mutants were prepared for Glu45, a residue close to the substrate entrance channel proposed to be involved in the termination of the cyclization of squalene. RESULTS: The DDTAVV motif in SHC was changed to DCTAEA, the corresponding conserved region of eukaryotic oxidosqualene cyclase (OSC), by the triple mutation of D377C/V380E/V381A; selected single mutants were also examined. The triple mutant showed no detectable cyclization of squalene, but effectively cyclized 2,3-oxidosqualene to give mono- and pentacyclic triterpene products. Of the Glu45 mutants, E45A and E45D showed reduced activity, E45Q showed slightly increased activity, and E45K was inactive. A normal yield of pentacyclic products was produced, but the ratio of hopene 2 to hopanol 3 was significantly changed in the less active mutants. CONCLUSIONS: Initiation and substrate selectivity may be determined by the interaction of the DDTAVV motif with the isopropylidene of squalene (for SHC) and of the DCTAEA motif with the epoxide of oxidosqualene (for OSC). This is the first report of a substrate switch determined by a central catalytic motif in a triterpenoid cyclase. At the termination of cyclization, the product ratio may be largely controlled by Glu45 at the entrance channel to the active site.     

Biosynthetic Mechanism of Lanosterol: Cyclization

The remarkable cyclization mechanism of the formation of the 6‐6‐6‐5 tetracyclic lanosterol (a key triterpenoid intermediate in the biosynthesis of cholesterol) from the acyclic 2,3‐oxidosqualene catalyzed by oxidosqualene cyclase (OSC) has stimulated the interest of chemists and biologists for over a half century. Herein, the elaborate, state‐of‐the‐art two‐dimensional (2D) QM/MM MD simulations have clearly shown that the cyclization of the A–C rings involves a nearly concerted, but highly asynchronous cyclization, to yield a stable intermediate with “6‐6‐5” rings followed by the ring expansion of the C‐ring concomitant with the formation of the D‐ring to yield the “6‐6‐6‐5” protosterol cation. The calculated reaction barrier of the rate‐limiting step (≈22 kcal mol^?1) is comparable to the experimental kinetic results. Furthermore all previous experimental mutagenic evidence is highly consistent with the identified reaction mechanism.     

Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis

Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and cycloartenol synthases, respectively. These results indicated the presence of distinct OSCs for cycloartenol and cucurbitadienol synthesis in this plant.     

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. We developed a simple method to evaluate the in vivo enzymatic activity; the expression levels of EtASs and the quantities of the cyclic triterpenes produced were determined by use of western blot and GC analyses, respectively. Measurement of the relative in vivo activity of the mutants versus that of the wild‐type enzyme showed that the Ala, Met, His, and Trp variants had significantly decreased activity, but that the Tyr mutant had a high activity, which was nearly the same as that of the wild‐type enzyme. In contrast to Tyr, Ala and Met possess no π‐electrons; thus, the role of Phe728 is to stabilize the cationic intermediates, resulting in facilitation of the ring‐expansion processes, especially by stabilizing the secondary cations. The decreased activity of the Trp mutant is ascribed to the introduction of a large steric bulk, leading to looser binding of oxidosqualene in the Trp variant. The His mutant afforded germanicol as the main product, indicating that the Phe residue is located near the D/E‐ring‐formation site. Changes in the steric bulk gave some cationic intermediates, resulting in the formation of 13 cyclic triterpenes, including an unnatural triterpene, (17E)‐dammara‐17(20),24‐dien‐3β‐ol, and isoursenol, which has rarely been found in nature. In this study, we provide the first experimental evidence that cation–π interactions play a key role in the catalytic action of OSCs.     

还原热处理对铈锆铝复合氧化物性能的影响

采用共沉淀法制备铈锆铝复合氧化物(CeO2-ZrO2-Al2O3,CZA)和铈锆复合氧化物(CeO2-ZrO2,CZ),将样品分别在空气和10%H2/Ar气氛下进行热处理,利用X射线粉末衍射(XRD)、O2脉冲吸附、H2程序升温还原(H2-TPR)等手段研究了复合氧化物的结构及性能.结果表明:CZA样品经950℃还原热处理后出现CeAlO3晶相,热处理温度越高,越有利于CeAlO3物相生成.CZA储氧量(OSC)随着还原热处理温度升高逐渐增大,至900℃达到1270.3μmo·lg-1;温度继续升高,OSC减小,1100℃还原热处理后CZA的OSC仅为23.2μmo·lg-1.研究发现还原热处理中形成CeAlO3,其显著影响CZA样品的储氧性能和还原性能.     

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β-amyrin, respectively.     

Arabidopsis thaliana LUP1 converts oxidosqualene to multiple triterpene alcohols and a triterpene diol

The Arabidopsis thaliana LUP1 gene encodes an enzyme that converts oxidosqualene to pentacyclic triterpenes. Lupeol and beta-amyrin were previously reported as LUP1 products. Further investigation described here uncovered the additional products germanicol, taraxasterol, psi-taraxasterol, and 3,20-dihydroxylupane. These results suggest that the 80 known C(30)H(50)O compounds that are structurally consistent with being oxidosqualene cyclase products may be derived from fewer than 80 enzymes and that some C(30)H(52)O(2) compounds may be direct cyclization products of oxidosqualene.     

An oxidosqualene cyclase makes numerous products by diverse mechanisms: a challenge to prevailing concepts of triterpene biosynthesis

The genome of the model plant Arabidopsis thaliana encodes 13 oxidosqualene cyclases, 9 of which have been characterized by heterologous expression in yeast. Here we describe another cyclase, baruol synthase (BARS1), which makes baruol (90%) and 22 minor products (0.02-3% each). This represents as many triterpenes as have been reported for all other Arabidopsis cyclases combined. By accessing an extraordinary repertoire of mechanistic pathways, BARS1 makes numerous skeletal types and deprotonates the carbocation intermediates at 14 different sites around rings A, B, C, D, and E. This undercurrent of structural and mechanistic diversity in a superficially accurate enzyme is incompatible with prevailing concepts of triterpene biosynthesis, which posit tight control over the mechanistic pathway through cation-pi interactions, with a single proton acceptor in a hydrophobic active site. Our findings suggest that mechanistic diversity is the default for triterpene biosynthesis and that product accuracy results from exclusion of alternative pathways.     

Degradation of the antiviral drug oseltamivir carboxylate in surface water samples

Numerous studies have documented that a wide number of pharmaceuticals used in human and veterinary medicine have the potential to enter the aquatic ecosystem. The antiviral prodrug oseltamivir phosphate has received recent attention with regard to its possible use against the highly pathogenic H5N1 virus. This preliminary laboratory study investigated the persistence of the active antiviral drug, oseltamivir carboxylate (OSC), in water samples taken from an irrigation canal. After an initial rapid decrease, OSC concentrations slowly decreased during the remaining incubation period. Approximately 65% of the initial OSC amount remained in water at the end of the 36-day incubation period. A small amount of OSC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role for microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OSC persistence. Presence of OSC (1.5?µg?mL^?1) did not significantly affect the metabolic potential of the water microbial population, estimated by glyphosate and metolachlor mineralization. In contrast, OSC caused an initial transient decrease in the size of the indigenous microbial population of water samples.     

Design,Synthesis, and Photovoltaic Characterization of a Small Molecular Acceptor with an Ultra‐Narrow Band Gap

The design of narrow band gap (NBG) donors or acceptors and their application in organic solar cells (OSCs) are of great importance in the conversion of solar photons to electrons. Limited by the inevitable energy loss from the optical band gap of the photovoltaic material to the open‐circuit voltage of the OSC device, the improvement of the power conversion efficiency (PCE) of NBG‐based OSCs faces great challenges. A novel acceptor–donor–acceptor structured non‐fullerene acceptor is reported with an ultra‐narrow band gap of 1.24 eV, which was achieved by an enhanced intramolecular charge transfer (ICT) effect. In the OSC device, despite a low energy loss of 0.509 eV, an impressive short‐circuit current density of 25.3 mA cm⁻² is still recorded, which is the highest value for all OSC devices. The high 10.9 % PCE of the NBG‐based OSC demonstrates that the design and application of ultra‐narrow materials have the potential to further improve the PCE of OSC devices.     

Design,Synthesis, and Photovoltaic Characterization of a Small Molecular Acceptor with an Ultra‐Narrow Band Gap

The design of narrow band gap (NBG) donors or acceptors and their application in organic solar cells (OSCs) are of great importance in the conversion of solar photons to electrons. Limited by the inevitable energy loss from the optical band gap of the photovoltaic material to the open‐circuit voltage of the OSC device, the improvement of the power conversion efficiency (PCE) of NBG‐based OSCs faces great challenges. A novel acceptor–donor–acceptor structured non‐fullerene acceptor is reported with an ultra‐narrow band gap of 1.24 eV, which was achieved by an enhanced intramolecular charge transfer (ICT) effect. In the OSC device, despite a low energy loss of 0.509 eV, an impressive short‐circuit current density of 25.3 mA cm⁻² is still recorded, which is the highest value for all OSC devices. The high 10.9 % PCE of the NBG‐based OSC demonstrates that the design and application of ultra‐narrow materials have the potential to further improve the PCE of OSC devices.     

Enzyme redesign: two mutations cooperate to convert cycloartenol synthase into an accurate lanosterol synthase

Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Described herein are experiments in which cycloartenol synthase was redesigned to become a highly accurate lanosterol synthase. Several single mutants have been described that modify the catalytic specificity of cycloartenol to form some lanosterol. Modeling studies were undertaken to identify combinations of mutations that cooperate to decrease the formation of products other than lanosterol. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation.     

Steric bulk at cycloartenol synthase position 481 influences cyclization and deprotonation

Cycloartenol synthase converts oxidosqualene to the pentacyclic sterol precursor cycloartenol. An Arabidopsis thaliana cycloartenol synthase Ile481Val mutant was previously shown to produce lanosterol and parkeol in addition to its native product cycloartenol. Experiments are described here to construct Phe, Leu, Ala, and Gly mutants at position 481 and to determine their cyclization product profiles. The Phe mutant was inactive, and the Leu mutant produced cycloartenol and parkeol. The Ala and Gly mutants formed lanosterol, cycloartenol, parkeol, achilleol A, and camelliol C. Monocycles comprise most of the Gly mutant product, showing that an alternate cyclization route can be made the major pathway by a single nonpolar mutation.     

晶格氧部分氧化甲烷制合成气

用储氧材料中的晶格氧代替分子氧部分氧化甲烷制合成气,并以空气、H₂O或CO₂为氧源对失去晶格氧的储氧材料进行氧化再生,是一种通过气固反应制取合成气的新工艺。具有较高经济效益和环境效益。本文综述了外该技术在储氧材料和反应体系等方面的研究进展,并从合成气和金属联产工艺以及熔融盐储能的研究思路中得出了一些启示,对该技术今后的研究重点和应用领域进行了展望。     

Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase: A chemistry–biology interdisciplinary study of the protein's structure–function–reaction mechanism relationships

The oxidosqualene cyclases (EC 5.4.99‐) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)‐2,3‐oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure–function relationships of the Saccharomyces cerevisiae oxidosqualene‐lanosterol cyclase‐catalyzed cyclization/rearrangement reaction. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 302–325; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20157     

The origin of the enhanced oxygen storage capacity of Ce(1-x)(Pd/Pt)(x)O2

Doping CeO(2) with Pd or Pt increases the oxygen storage capacity (OSC) and catalytic activity of this environmentally important material. To date, however, an understanding of the mechanism underlying this improvement has been lacking. We present a density functional theory analysis of Pd- and Pt-doped CeO(2), and demonstrate that the increased OSC is due to a large displacement of the dopant ions from the Ce lattice site. Pd(II)/Pt(II) (in a d(8) configuration) moves by ～1.2 ? to adopt a square-planar coordination due to crystal field effects. This leaves three three-coordinate oxygen atoms that are easier to remove, and which are the source of the increased OSC. These results highlight the importance of rationalizing the preferred coordination environments of both dopants and host cations when choosing suitable dopants for next generation catalysts.