| Carbohydrates are an important class of organic compounds which widely exist in nature.They are not only the main energy source for all life activities,but also participate in many crucial biological processes as cell structural molecules.Carbohydrates can be conjugated to polypeptides,proteins,and lipids in a process called glycosylation to form glycopeptides,glycoproteins,and glycolipids respectively,and participate in cell recognition,cell migration,inflammation as well as infection caused by bacteria and viruses.For diversified natural products with bioactivities,glycosylation alters their architectures and endows them with new activies,thus their synthesis has attacted great attentionA prerequisite for study of these glycosylated natural products is to adequately synthesize them,which still faces great challenges.Chemical synthesis and biological enzymatic synthesis are two main ways to synthesize carbohydrates in vitro.The advantage of chemical synthesis lies in the flexibility of its synthesis route,while it is accompanied by poor selectivity.Besides,the chemical synthetic process involves cumbersome protection and deprotection steps for key groups,which may lead to low yield and waste of products,and it is also difficult to synthesize special glycosidic bonds,such as ?-glycosidic bonds.As an effective alternative to chemical synthesis,the biological enzymatic method has been used to synthesize a variety of oligosaccharides,polysaccharides,and glycoconjugates due to its mild reaction conditions and strong selectivity.Glycosidases and glycosyltransferases are currently the two main tools for the synthesis of carbohydrates.Glycosyltransferases(EC 2.4.x.y)catalyze the transfer of active glycosyl groups to acceptor molecules,such as lipids,oligosaccharides,proteins,and nucleic acids.Glycosyltransferases have attracted much attention due to their strict stereoselectivity and regioselectivity,high catalytic efficiency,and specific products.Rhamnosyltransferases(Rha-Ts,EC 2.4.1.159)are a typical class of glycosyltransferases which widely exist in bacteria and plants and catalyze the synthesis of rhamnosylated compounds.Cancer has superseded heart disease as the main cause of death worldwide.The current clinical treatments for cancer are mainly surgery,radiotherapy,and chemotherapy,the latter two of which damage normal cells and destroy the immune system.Some natural products,such as pterostilbene and resveratrol,show antiproliferative activities against cancer cells but little side effects on normal cells.Glycosylation can effectively improve the physicochemical properties and biological activities of natural products.Many natural rhamnosides show remarkable pharmacological activities,which were proved to be correlated with rhamnose modification.For example,the rhamnose binding lectins(RBLs)was revealed to be abundantly present on the surface of some human tumor cells,which preferentially bond to the rhamnosides of solasodine compared to the aglycone.Besides,rhamnosylation can also stablize the modified compounds when they are administrated as drugs because no rhamnosidase has been found in human.Our group previously reported an ?-L-rhamnosidase RhaL1 from Alternaria sp.L1,which could catalyze the rhamnosylation of several anti-tumor drugs through reverse hydrolytic action.However,the products yields were low and the compounds used were limited to arficial molecules.Here in this study,we aimed to provide a detailed characterization of a rhamnosyltransferase newly found from Saccharopolyspora erythraea,which proved to be a versatile enzyme tool by rhamnosylating mulitple natural products.We also attempted to synthesize the novel rhamnoside deriatives of some bioactive natural products with this enzyme and evaluate the altered activities of the products,especially the effects on the proliferation of some human tumor cell lines.A rhamnosyltransferase Ses60310(WP015103398.1,gene sequence 1.1 kb,theoretical molecular weight 39.4 kDa)from Saccharothrix espanaensis was reported.Ses60310 belonged to GT1 family and could rhamnosylate quercetin,neogenic acid,and a variety of anthraquinone compounds with dTDP-L-rhamnose as the sugar donor.The broad aglycone spectrum of Ses60310 was considered to be attributed to its possible role in defending the host strain against the toxic natural products produced by other organisms through glycosylation of them.In this paper,the predicted rhamnosyltransferase gene SeRhaT(GenBank accession No.CP069353.1)from Saccharopolyspora erythraea was found by BLAST using the amino acid sequence of Ses60310.The sequence identity between Ses60310 and SeRhaT was 55%.The gene SeRhaT was cloned into pET-22b plasmid and overexpressed in E.coil BL21(DE3).The recombinant SeRhaT could use dTDP-L-rhamnose as the sugar donor to rhamnosylate totally eight natural products,including kaempferol,honokiol,magnolol,resveratrol,pterostilbene,esculin,genistein,and phloretin.Among all the positive aglycones,resveratrol and pterostilbene were rhamnosylated by SeRhaT with relatively higher yields according to the TLC result.Moreover,it was reported that they both have good anti-cancer activities.Therefore,we descided to synthesize the rhamnosides of resveratrol and pterostilbene and study their properties in detail.The optimal rhamnosylation conditions of SeRhaT,including optimal pH,temperature,metal ions,and substrate concentration,were explored with pterostilbene and dTDP-Rha.In the HPLC analysis,the product peak with a retention time of 10.6 min was found,and the ion peak with[M+H]+403.1751 was found in the mass spectrometry detection of the reaction solution,which is consistent with the theoretical molecular weight of pterostilbene rhamnoside(Pte-Rha)403.17.The results showed that the optimum reaction conditions to synthesize pterostilbene rhamnoside were as follows:2 mM pterostilbene,2 mM dTDP-Rha,8 mM Mg2+,pH 10,temperature 35?,time 16 h,and the maximum conversion was 50.6%.At the same time,it was found that metal ions affected the activity of SeRhaT.Mg2+,Ba2+,Mn2+,Zn2+,and other metal ions could increase the enzyme activity.Mg2+ was the best for SeRhaT catalysis,which could double SeRhaT activity.SeRhaT was stable within a wide range of temperature.It maintained relatively high activity at 4-15?.However,the enzyme activity of the enzyme will decrease rapidly at 25? and 35 ?,and it completely lost the activity at 45?.The enzymes showed a strong activity in neutral and weakly alkaline environment but its lost the activity at pH<4.0.The Km value of SeRhaT for dTDP-rhamnose was 595.81 ?M and the kcat value was 0.23 min-1;the Km value of SeRhaT for pterostilbene was 894.82 ?M,and the kcat value was 0.21 min-1.The rhamnoside of resveratrol was synthesized under the same conditions.In the HPLC detection of the reaction solution,the product peaks were found at 2 min,2.5 min,3.5 min and 4 min,and the ion peak with[M+H]+375.1413 was found in the mass spectrometry detection of the reaction solution,which was consistent with the theoretical molecular weight of resveratrol rhamnoside(Res-Rha)374.14.A reaction of 4 mL was used to synthesize the resveratrol rhamnoside in large scale with all the reactants added at the same concentrations with that used in the 20 ?L reaction.Under the optimal conditions,the maximal conversion of pterostilbene and resveratrol rhamnoside were 46%and 44%,respectively,and the yields were 0.37 mg/mL and 0.33 mg/mL,respectively.The rhamnoside products of pterostilbene and resveratrol were tried to be purified using HPLC.However,the presence of multiple hdyroxyls in resveratrol led to several product isomers formed in the rhamnosylation mediated by SeRhaT.These products could not be thoroughly separated in HPLC,hampering the isolation of pure resveratrol rhamnosides.In contrast,pterostilbene had only one free hydroxyl which could be rhamnosylated by SeRhaT,thus the follow-up experiments centered on pterostilbene rhamnoside,which was more easily for preparation.The anticancer activity of pterostilbene rhamnoside was then evaluated in comparison with pterostilbene.The results showed that pterostilbene had little toxicity to human breast cancer cell line MCF-7,human normal breast cell line MCF-10A,human hepatoma cell line HepG2,and human normal liver cell line L-02.However,under the same conditions,pterostilbene rhamnoside had a certain inhibitory effect on all of tested cancer cells.The inhibitory effect on HepG2 which was reported to express more RBLs on cell surface was the most strong.The proliferation of MCF-10A and L-02 cells was inhibited at 48 h.Pterostilbene had a certain inhibitory effect on SW480 cells,and the rhamnosylated deriative showed an enhanced cytotoxicity.In summary,we characterized a novel rhamnosyltransferase SeRhaT from S.erythraea and found it could rhamnosylate a variety of natural products with dTDP-L-rhamnose as the sugar donor.Using SeRhaT,we successfully synthesized the rhamnoside of pterostilbene in quantity and studied its antiproliferative activity on a series of human tumor cells.This work provided a new enzyme for natural products rhamnosylation and also a novel promising leading compound. |
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