Functional Characterization of a Regiospecific Sugar-O-Methyltransferase from Nocardia.

Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. IMPORTANCE ThnM1 is a putative sugar-O-methyltransferase produced by the Nocardia sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.

Emodin 8-O-glucoside primes macrophages more strongly than emodin aglycone via activation of phagocytic activity and TLR-2/MAPK/NF-κB signalling pathway.

Emodin (Emo) is a natural plant anthraquinone derivative with a wide spectrum of pharmacological properties, including anticancer, antioxidant, and hepatoprotective activities. Glycosylation of natural anthraquinones with various sugar moieties can affect their physical, chemical, and biological functions. In this study, the potential immunomodulatory activities of Emo and its glycosylated derivative, emodin 8-O-glucoside (E8G), were evaluated and compared using murine macrophage RAW264.7 cells and human monocytic THP-1 cells. The results showed that E8G (20 µM) induced the secretion of TNF-α and IL-6 from RAW264.7 cells more effectively than unglycosylated Emo aglycone, by 4.9- and 1.6-fold, respectively, with no significant cytotoxicity in the concentration range tested (up to 20 µM). E8G (2.5-20 µM) significantly and dose-dependently induced inducible nitric oxide synthase (iNOS) expression by up to 3.2-fold compared to that of untreated control following a remarkable increase in nitric oxide (NO) production. E8G also significantly increased the expression of TLR-2 mRNA and the phosphorylation of MAPKs (JNK and p38). The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with E8G (2.5-20 µM). Moreover, E8G markedly induced macrophage-mediated phagocytosis of apoptotic Jurkat T cells. These results demonstrated that E8G far more strongly stimulates the secretion of proinflammatory cytokines, such as TNF-α and IL-6, and NO production from macrophages through upregulation of the TLR-2/MAPK/NF-κB signalling pathway than its nonglycosylated form, Emo aglycone. These results suggest for the first time that E8G may represent a novel immunomodulator, enhancing the early innate immunity.

Regio-specific biotransformation of alizarin to alizarin methoxide with enhanced cytotoxicity against proliferative cells.

Alizarin has been reported to have an antigenotoxic activity along with an inhibitory effect on the tumor cell growth of human colon carcinoma cells. Alizarin was biotransformed into an O-methoxide derivative using O-methyltransferase from Streptomyces avermitilis MA4680 (SaOMT2) to enhance its bioefficacy. The biotransformed product was extracted, purified, and characterized using various chromatographic and spectroscopic analyses, and confirmed to be an alizarin 2-O-methoxide. The antiproliferative activity of the compound against gastric cancer cells (AGS), uterine cervical cancer (Hela), liver cancer (HepG2), and normal cell lines was investigated. Alizarin 2-O-methoxide showed an inhibitory effect on all three cancer-cell lines at very low concentrations, from 0.078 µM, with no cytotoxicity against 267B1 (human prostate epithelial) and MRC-5 (normal human fetal lung fibroblast).

Biosynthesis of Rhamnosylated Anthraquinones in Escherichia coli.

Rhamnose is a naturally occurring deoxysugar present as a glycogenic component of plant and microbial natural products. A recombinant mutant Escherichia coli strain was developed by overexpressing genes involved in the TDP-L-rhamnose biosynthesis pathway of different bacterial strains and Saccharothrix espanaensis rhamnosyl transferase to conjugate intrinsic cytosolic TDP-L-rhamnose with anthraquinones supplemented exogenously. Among the five anthraquinones (alizarin, emodin, chrysazin, anthrarufin, and quinizarin) tested, quinizarin was biotransformed into a rhamoside derivative with the highest conversion ratio by whole cells of engineered E. coli. The quinizarin glycoside was identified by various chromatographic and spectroscopic analyses. The anti-proliferative property of the newly synthesized rhamnoside, quinizarin-4-O-α-L-rhamnoside, was assayed in various cancer cells.

Microbial Synthesis of Non-Natural Anthraquinone Glucosides Displaying Superior Antiproliferative Properties.

Anthraquinones, naturally occurring bioactive compounds, have been reported to exhibit various biological activities, including anti-inflammatory, antiviral, antimicrobial, and anticancer effects. In this study, we biotransformed three selected anthraquinones into their novel O-glucoside derivatives, expressing a versatile glycosyltransferase (YjiC) from Bacillus licheniformis DSM 13 in Escherichia coli. Anthraflavic acid, alizarin, and 2-amino-3-hydroxyanthraquinone were exogenously fed to recombinant E. coli as substrate for biotransformation. The products anthraflavic acid-O-glucoside, alizarin 2-O-ß-d-glucoside, and 2-amino-3-O-glucosyl anthraquinone produced in the culture broths were characterized by various chromatographic and spectroscopic analyses. The comparative anti-proliferative assay against various cancer cells (gastric cancer-AGS, uterine cervical cancer-HeLa, and liver cancer-HepG2) were remarkable, since the synthesized glucoside compounds showed more than 60% of cell growth inhibition at concentrations ranging from ~50 µM to 100 µM. Importantly, one of the synthesized glucoside derivatives, alizarin 2-O-glucoside inhibited more than 90% of cell growth in all the cancer cell lines tested.