Endogenous salicylic acid shows different correlation with baicalin and baicalein in the medicinal plant Scutellaria baicalensis Georgi subjected to stress and exogenous salicylic acid.

Salicylic acid (SA) is synthesized via the phenylalanine lyase (PAL) and isochorismate synthase (ICS) pathways and can influence the stress response in plants by regulating certain secondary metabolites. However, the association between SA and particular secondary metabolites in the Chinese medicinal plant Scutellaria baicalensis Georgi is unclear. To elucidate the association between SA and the secondary metabolites baicalin and baicalein, which constitute the primary effective components of S. baicalensis, we subjected seedlings to drought and salt stress and exogenous SA treatment in a laboratory setting and tested the expression of PAL and ICS, as well as the content of free SA (FSA), total SA (TSA), baicalin, and baicalein. We also assessed the correlation of FSA and TSA with PAL and ICS, and with baicalin and baicalein accumulation, respectively. The results indicated that both FSA and TSA were positively correlated with PAL, ICS, and baicalin, but negatively correlated with baicalein. The findings of this study improve our understanding of the manner in which SA regulates secondary metabolites in S. baicalensis.

Design strategies of oxidosqualene cyclase inhibitors: Targeting the sterol biosynthetic pathway.

Targeting the sterol biosynthesis pathway has been explored for the development of new bioactive compounds. Among the enzymes of this pathway, oxidosqualene cyclase (OSC) which catalyzes lanosterol cyclization from 2,3-oxidosqualene has emerged as an attractive target. In this work, we reviewed the most promising OSC inhibitors from different organisms and their potential for the development of new antiparasitic, antifungal, hypocholesterolemic and anticancer drugs. Different strategies have been adopted for the discovery of new OSC inhibitors, such as structural modifications of the natural substrate or the reaction intermediates, the use of the enzyme's structural information to discover compounds with novel chemotypes, modifications of known inhibitors and the use of molecular modeling techniques such as docking and virtual screening to search for new inhibitors. This review brings new perspectives on structural insights of OSC from different organisms and reveals the broad structural diversity of OSC inhibitors which may help evidence lead compounds for further investigations with various therapeutic applications.

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.

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents.

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

Characterisation of two oxidosqualene cyclases responsible for triterpenoid biosynthesis in Ilex asprella.

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and ß-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises ß-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.

Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, ß-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications.

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including ß-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

[Cloning and bioinformatics analysis of cycloartenol synthase (HcCAS1) gene in Huperzia carinata].

OBJECTIVE: To clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata. METHOD: After searching the transcriptome dataset of H. carinata, one unique sequence containing oxide squalene cyclases domain was discovered. The primers were designed according to the cDNA sequence of CAS from the dataset. And then, the open reading frame of CAS was cloned by RT-PCR strategy with the template of mixed RNA extracted from root, stem and leaf of H. carinata. The bioinformatic analysis of this gene and its corresponding protein was performed. RESULT: One unique sequence of CAS, named as HcCAS1 (GenBank accession number JN790125) , was cloned from H. carinata. The open reading frame of HcCAS1 consists of 2 474 bp, encoding one polypeptide with 757 amino acids. CONCLUSION: This study cloned and analyzed CAS from H. carinata for the first time. The result will provide a foundation for exploring the mechanism of sterol biosynthesis in Huperziaceae plants.

Cloning and characterization of a ß-amyrin synthase gene from the medicinal tree Aralia elata (Araliaceae).

Aralia elata is an important medicinal plant in China; it produces large amounts of oleanane type triterpene saponins. A full-length cDNA encoding ß-amyrin synthase (designated as AeAS) was isolated from young leaves of A. elata by reverse transcription-PCR. The full-length cDNA of AeAS was found to have a 2292-bp open reading frame, encoding a protein with 763 amino acid residues. The deduced amino acid sequence of AeAS showed the highest identity (97%) to Panax ginseng ß-amyrin synthase. When AeAS cDNA was expressed in Escherichia coli, an 87.8-kDa recombinant protein was detected by SDS-PAGE and Western blotting. The sequence was also heterologously expressed in the yeast Pichia pastoris, and production of ß-amyrin was detected by HPLC. Tissue expression pattern analysis by real-time reverse transcription-PCR revealed that AeAS is strongly expressed in leaves and stems, and weakly expressed in roots and flowers.

Molecular cloning and characterization of a tocopherol cyclase gene from Lactuca sativa (Asteraceae).

Tocopherol cyclase is a rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding tocopherol cyclase (designated as LsTC) was cloned from lettuce (Lactuca sativa) for the first time by rapid amplification of cDNA ends (RACE) and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTC was 1675 bp, with an open reading frame of 1521 bp, encoding a tocopherol cyclase protein of 506 amino acids, with a calculated molecular mass of 56.76 kD and an isoelectric point of 6.49. Comparative analysis revealed that LsTC has a close similarity with tocopherol cyclases from other plant species. Bioinformatic analysis indicated that LsTC shares a common evolutionary origin based on sequence and has the closest relationship to tocopherol cyclase from Helianthus annuus. Quantitative RT-PCR analysis suggested that expression of LsTC is induced and strengthened by oxidative stresses, such as strong light and drought. This cloning and characterization of LsTC will be helpful for further understanding of its role in the tocopherol biosynthesis pathway and provide a candidate gene for metabolic engineering of vitamin E.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

[Impacting of polymorphism of beta-amyrin synthase from Glycyrrhiza uralensis on its catalytic efficiency].

OBJECTIVE: To analyze heterologous expression in Saccharomyces cerevisiae of two genotypes: beta-AS (A-T) genotype which is related to high content of glycyrrhizic acid and beta-AS(G-C) genotype which is related to low content of glycyrrhizic acid, and compare two different genotypes on the impact of beta-amyrin production in order to provide a foundation for licorice molecular breeding. METHOD: The 2 289 bp fragment in plasmid pMD-19T encoding beta-amyrin synthase was subcloned into the yeast-Escherichia coli shuttle vector pY26, thus an expression recombinant plasmid PY-beta-AS containing target gene was constructed. The PY-beta-AS was introduced into defective mutant INVSc1 of S. cerevisiae by LiAc method, after induced by IPTG, the content of beta-amyrin was determined by GC-MS. RESULT: GC-MS analysis demonstrates that the an occurring peak corresponding to beta-amyrin standards was detected with the same retention time, which is absent in the cell transform with empty vector. Results showed the peak was beta-amyrin and the percentage of beta-amyrin in two genotypes: beta-AS (A-T) genotype and beta-AS (G-C) genotype were 19.08% and 1.40%, respectively. Thus the beta-amyrin synthase exhibited the activity of catalyzing 2, 3-oxidosqualene to beta-amyrin. CONCLUSION: The catalytic efficiency of beta-AS(A-T) genotype is higher than that of beta-AS(G-C) genotype, which can lay the foundation for licorice molecular breeding.

Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua.

Using a degenerate primer designed from triterpene synthase sequences, we have isolated a new gene from the medicinal plant Artemisia annua. The predicted protein is highly similar to beta-amyrin synthases (EC 5.4.99.-), sharing amino acid sequence identities of up to 86%. Expression of the gene, designated AaBAS, in Saccharomyces cerevisiae, followed by GC/MS analysis, confirmed the encoded enzyme as a beta-amyrin synthase. Through engineering the sterol pathway in S. cerevisiae, we explore strategies for increasing triterpene production, using AaBAS as a test case. By manipulation of two key enzymes in the pathway, 3-hydroxy-3-methylglutaryl-CoA reductase and lanosterol synthase, we have improved beta-amyrin production by 50%, achieving levels of 6 mg.L(-1) culture. As we have observed a 12-fold increase in squalene levels, it appears that this strain has the capacity for even higher beta-amyrin production. Options for further engineering efforts are explored.

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.

A catalytic antibody against a tocopherol cyclase inhibitor.

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.

Structure of a cephalosporin synthase.

Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (alpha-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.