Exploring the catalytic diversity of two short-chain dehydrogenases/reductases from Stachybotrys chartarum.

Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize ß-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Enzymatic synthesis of anhydroicaritin, baohuoside and icariin.

Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue.

Two new 5/6/6 polyketide-amino acid hybrids from a genetic mutant of Periconia sp. F-31.

Peridecalins C and D (1 and 2), one decalin and one oxygen-decalin containing polyketide-amino acid hybrids with 5/6/6 ring system, was isolated from a genetic mutant of Periconia sp. F-31. Their structures were elucidated through extensive spectroscopic data analysis, including 1 D/2D NMR and HR-MS spectra. Biosynthetically, two proposed Diels-Alder reactions are supposed to be involved in the skeleton construction of 1 and 2.

Sesquiterpenes from the endophytic fungus Periconia sp. F-31.

One new eremophilane sesquiterpene periconianone L (1), together with four known guaiane-type sesquiterpenes 4,10,11-trihydroxyguaiane (2), (-)-guai-1(10)-ene-4α,11-diolhydroxymecuration (3), guaidiol A (4), and epi-guaidiol A (5) were isolated from the endophytic fungus Periconia sp. F-31. The structure of the new compound was established by spectroscopic methods, including UV, IR, HRESIMS, and extensive NMR techniques. Compound 3 was isolated as natural product for the first time.

Biosynthesis of polyketides by two type III polyketide synthases from Aloe barbadensis.

Various bioactive polyketides have been found in Aloe barbadensis. However, the polyketide synthases (PKSs), which participate in biosynthesis of polyketides in A. barbadensis remain unknown. In this study, two type III PKSs (AbPKS1 and AbPKS2) were identified from A. barbadensis. AbPKS1 and AbPKS2 were able to utilize malonyl-CoA to yield heptaketides (TW93a and aloesone) and octaketides (SEK4 and SEK4b), respectively. AbPKS1 also exhibited catalytic promiscuity in recognizing CoA thioesters of aromatics to produce unusual polyketides. What Is more, a whole cell biocatalysis system with the capability of producing 26.4 mg/L of SEK4/SEK4b and 2.1 mg/L of aloesone was successfully established.

Bistachybotrysin K, one new phenylspirodrimane dimer from Stachybotrys chartarum with potent cytotoxic activity.

Bistachybotrysin K (1), one new phenylspirodrimane dimer with a central 6/7 oxygen heterocycle core, was isolated from the fungus Stachybotrys chartarum CGMCC 3.5365. Its structure was elucidated by extensive spectroscopic data and single-crystal X-ray diffraction. Compound 1 showed significant cytotoxicity against human tumor cell lines HCT116, NCI-H460, BGC823, Daoy, and HepG2 with IC50 values in the range of 1.1-4.7 µM.

Three new phenylspirodrimane derivatives with inhibitory effect towards potassium channel Kv1.3 from the fungus Stachybotrys chartarum.

Three new phenylspirodrimanes derivatives named stachybotrysins H and I (1 and 2) and stachybotrin E (3), together with one known compound stachybotrylactam (4), were isolated from Stachybotrys chartarum CGMCC 3.5365. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 1 and 2 showed inhibitory effect towards potassium channel Kv1.3 with IC50 values of 13.4 and 10.9 µM, respectively.

Enzymatic synthesis of glucuronidated metabolites of two neurological active agents using plant glucuronosyltransferases.

Glucuronidation is an important and popular metabolic reaction in vivo of drugs. The further evaluation of biological activity and toxicity of glucuronides is necessary in the course of the drug research and development. However, the synthesis of glucuronides is limited by the lack of efficient approach. Herein, we have developed a new glucuronide synthesis method using plant uridine diphosphate-dependent glucuronosyltransferases (UGTs), UGT88D4, UGT88D7, and EpGT8, enabling the convenient preparation for corresponding O-glucuronide metabolites (1a, 2a, 3a, and 3b) in milligram scale of two neurological active agents, IMM-H004 (1) and FLZ (2). Their structures were characterized by spectroscopic data analyses.

Three xanthone dimers from the Thai mangrove endophytic fungus Phomopsis sp. xy21.

Three new xanthone dimers, named phomoxanthones C-E (1-3), were obtained from the Thai mangrove fungus Phomopsis sp. xy21, together with four known ones. The structures of these compounds were elucidated by the analysis of HRESIMS and extensive NMR spectroscopic data. The absolute configuration of 1 was established by the analysis of single-crystal X-ray diffraction with Cu Kα radiation. Phomoxanthones C (1) and D (2) possess a highly oxidized hexahydroxanthone skeleton.

Lanostane triterpenoids and ergostane-type steroids from the cultured mycelia of Ganoderma capense.

Two new lanostane triterpenoids (1 and 2), two new ergostane-type steroids (3 and 4) together with two known lanostane triterpenoids (5 and 6) and one known steroid (7) were isolated from the cultured mycelia of Ganoderma capense (CGMCC 5.71). Their structures were determined on the basis of extensive spectroscopic (HRESIMS, 1D NMR, 2D NMR) data analyses. Compound 1 exhibited moderate cytotoxic activity against the human cancer cell line NCI-H1650 with an IC50 value of 22.3 µM, and 7 displayed cytotoxic activity against the human cancer cell line HCT116 with an IC50 value of 17.4 µM. In addition, compounds 2, 3, 5, and 6 displayed weak anti-HIV activity with IC50 values of 23.5, 46.7, 21.6, and 30.1 µM, respectively.

Bioactive steroids and sorbicillinoids isolated from the endophytic fungus Trichoderma sp. Xy24.

A new steroid glucoside (1), along with nine known steroids (2-10) and four known sorbicillinoids (11-14), were isolated from the endophytic fungus Trichoderma sp. Xy24. Their structures were elucidated on the basis of spectroscopic data analyses and by comparison with reported data. Compounds 3, 5-7, 9, 10, and 13 exhibited significant inhibitory effects on HIV-1 virus with IC50 values ranging 1.9-9.3 µM; compounds 10, 13, and 14 showed potent inhibitory activity on LPS-induced NO production in BV2 microglia cells with inhibitory rates of 108.2, 100, and 75.1% at 10 µM, respectively. In addition, compound 10 displayed moderate cytotoxicity against BCG823 and HePG2 cell lines with IC50 values of 11.1 and 17.7 µM, respectively.

Neural anti-inflammatory sesquiterpenoids from the endophytic fungus Trichoderma sp. Xy24.

Three new sesquiterpenoids trichoacorenols B-C and cyclonerodiol B (1-3), along with three known ones (4-6), were isolated from the mangrove plant endophytic fungus Trichoderma sp. Xy24 using various column chromatography techniques. The structures of these compounds were determined on the basis of extensive spectroscopic data analyses. Compounds 1, 2, 4, and 5 were four acorane sesquiterpenes, 3 and 6 were two monocyclic sesquiterpenediols. Compounds 3 and 5 exhibited significant neural anti-inflammatory activity by inhibiting LPS-induced NO production in BV2 cells with the inhibitory rates of 75.0% and 39.2% at 0.1 µM, respectively, which are more potent than curcumin, a positive control with the inhibitory rate of 21.1% at 0.1 µM.

Four new monoterpenoids from an endophytic fungus Periconia sp. F-31.

Five monoterpenoids were isolated from the endophytic fungus Periconia sp. F-31, including three new carene-type monoterpenoids, 2-carene-5,8-diol (1), 2-carene-8,10-diol (2), 2-carene-8-acetamide (3), one new menthene-type monoterpenoid 8-hydroxy-1,7-expoxy-2-menthene (4), and one known monoterpenoid anethofuran (5). The structures of all compounds were elucidated based on a comprehensive spectroscopic data analysis, electronic circular dichroism (ECD), and calculated ECD.

[Enzymatic glycosylation of 4'-demethylepipodophyllotoxin].

The glycosides of 4'-demethylepipodophyllotoxin (DMEP) possess various pharmacological activities; however, the chemical synthesis of these glycosides faces challenges in regioselectivity, stereoselectivity, and the protection and de-protection of functional groups. In this work, a novel glycosyltransferase (GT) gene AbGT5 from Aloe barbadensis was successfully cloned, heterogeneously expressed and purified. Recombinant AbGT5 was able to catalyze the glycosylation of DMEP and the glycosylated product, which was separated from the preparative scale reaction, was characterized as DMEP 4'-O-ß-D-glucoside via MS, 1H-NMR, 13C-NMR, HSQC and HMBC. According to the investigations of enzyme properties, AbGT5 show the highest activity around 20 ℃ in the buffer of pH 9.0, and it was independent of divalent metal ions. Under the optimum conditions, the conversion rate of DMEP can reach 80%. Above all, in this work the enzymatic glycosylation of DMEP was achieved with high efficiency by the novel GT AbGT5.

Flavonoids from the cultured cells of Glycyrrhiza uralensis.

Two new flavonoids (1 and 2), along with 14 known ones (3-16), were isolated from the cultured cells of Glycyrrhiza uralensis. Most of them were prenylated flavonoids. Their structures were elucidated on the basis of spectroscopic data analysis. All compounds showed non-cytotoxicity against five human tumor cell lines. The results suggest that plant cultured cells can yield the secondary metabolites that have not been found in parent plant.

[Cloning,expression and functional identification of secoisolariciresinol dehydrogenase gene from Dysosma versipellis callus].

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin.

[Flavonoid glycosides from callus cultures of Dysosma versipellis].

Various chromatographic techniques, including silica gel column chromatography, Sephadex LH-20, preparative thin-layer chromatography, and preparative HPLC, were employed to isolate the chemical constituents from callus cultures of Dysosma versipellis. Structures of the compounds were elucidated based on UV, IR, MS and NMR spectroscopic data analysis. Totally, seven flavonoid glycosides were isolated from the 95% ethanol extract of the callus cultures and identified as kaempferol-3-O-[6″-(3″'-methoxy)-malonyl]-ß-D-glucopyranoside(1), kaempferol-3-O-(6″-O-acetyl)-ß-D-glucopyranoside(2), kaempferide-3-O-ß-D-glucopyranoside(3), kaempferol-3-O-ß-D-glucopyranoside(4), isoquercitrin(5), quercetin-4'-O-ß-D-glucopyranoside(6) and kaempferol-3-(6″-malonyl)-ß-D-glucopyranoside(7), respectively.All these compounds were isolated from callus cultures of D. versipellis for the first time.Compounds 1, 2, 3, 6 and 7 were firstly obtained from plant materials of D. versipellis, and compound 1 was a new compound.

New 2-arylbenzofuran metabolite from cell cultures of Morus alba.

A new 2-arylbenzofuran compound, 5-dehydroxy-moracin U (1), along with 10 known compounds (2-11), were isolated from cell cultures of Morus alba. Their structures were elucidated on the basis of extensive spectroscopic analyses. The anti-inflammatory activity assay of 1-8 showed that 2 and 8 exhibited significant inhibitory effect on LPS-induced NO production with the values of 76.4% and 98.7% at 10(- 5) M, respectively.

Regio-selective deglycosylation of icariin by cell suspension cultures of Glycyrrhiza uralensis and Morus alba.

Biotransformations of icariin (1) by cell suspension cultures of Glycyrrhiza uralensis and Morus alba yielded two new metabolites, icaruralins A and B (2 and 3), and one known metabolite, baohuoside I (4). Their structures were determined on the basis of extensive spectroscopic analysis. This is the first report that the cell suspension cultures of G. uralensis and M. alba possess deglycosylation functionality.