C-glycosyltransferases Research Articles

A highly selective C-rhamnosyltransferase from Viola tricolor and insights into its mechanisms.

C-Glycosides are important natural products with various bioactivities. In plant biosynthetic pathways, the C-glycosylation step is usually catalyzed by C-glycosyltransferases (CGTs), and most of them prefer to accept uridine 5'-diphosphate glucose (UDP-Glc) as sugar donor. No CGTs favoring UDP-rhamnose (UDP-Rha) as sugar donor has been reported, thus far. Herein, we report the first selective C-rhamnosyltransferase VtCGTc from the medicinal plant Viola tricolor. VtCGTc could efficiently catalyze C-rhamnosylation of 2-hydroxynaringenin 3-C-glucoside, and exhibited high selectivity towards UDP-Rha. Mechanisms for the sugar donor selectivity of VtCGTc were investigated by molecular dynamics (MD) simulations and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) binding free energy calculations. Val144 played a vital role in recognizing UDP-Rha, and the V144T mutant could efficiently utilize UDP-Glc. This work provides a new and efficient approach to prepare flavonoid C-rhamnosides such as violanthin and iso-violanthin.

Sequence mining yields 18 phloretin C-glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin-di-C-glycoside.

C-glycosyltransferases (C-GTs) offer selective and efficient synthesis of natural product C-glycosides under mild reaction conditions. In contrast, the chemical synthesis of these C-glycosides is challenging and environmentally harmful. The rare occurrence of C-glycosylated compounds in Nature, despite their stability, suggests that their biosynthetic enzymes, C-GTs, might be scarce. Indeed, the number of characterized C-GTs is remarkably lower than O-GTs. Therefore, discovery efforts are crucial for expanding our knowledge of these enzymes and their efficient application in biocatalytic processes. This study aimed to identify new C-GTs based on their primary sequence. 18 new C-GTs were discovered, 10 of which yielded full conversion of phloretin to its glucosides. Phloretin is a dihydrochalcone natural product, with its mono-C-glucoside, nothofagin, having various health-promoting effects. Several of these enzymes enabled highly selective production of either nothofagin (UGT708A60 and UGT708F2) or phloretin-di-C-glycoside (UGT708D9 and UGT708B8). Molecular docking simulations, based on structural models of selected enzymes, showed productive binding modes for the best phloretin C-GTs, UGT708F2 and UGT708A60. Moreover, we characterized UGT708A60 as a highly efficient phloretin mono-C glycosyltransferase (kcat =2.97 s-1 , KM =0.1μM) active in non-buffered, dilute sodium hydroxide (0.1-1mM). We further investigated UGT708A60 as an efficient biocatalyst for the bioproduction of nothofagin.

Identification of a flavonoid C-glycosyltransferase from fern species Stenoloma chusanum and the application in synthesizing flavonoid C-glycosides in Escherichia coli

BackgroundFlavonoid C-glycosides have many beneficial effects and are widely used in food and medicine. However, plants contain a limited number of flavonoid C-glycosides, and it is challenging to create these substances chemically.ResultsTo screen more robust C-glycosyltransferases (CGTs) for the biosynthesis of flavonoid C-glycosides, one CGT enzyme from Stenoloma chusanum (ScCGT1) was characterized. Biochemical analyses revealed that ScCGT1 showed the C-glycosylation activity for phloretin, 2-hydroxynaringenin, and 2-hydroxyeriodictyol. Structure modeling and mutagenesis experiments indicated that the glycosylation of ScCGT1 may be initiated by the synergistic action of conserved residue His26 and Asp14. The P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor. Furthermore, when using phloretin as a substrate, the extracellular nothofagin production obtained from the Escherichia coli strain ScCGT1-P164T reached 38 mg/L, which was 2.3-fold higher than that of the wild-type strain. Finally, it is proved that the coupling catalysis of CjFNS I/F2H and ScCGT1-P164T could convert naringenin into vitexin and isovitexin.ConclusionThis is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering.

Identification and characterization of glycosyltransferases catalyzing direct xanthone 4-C-glycosylation in Hypericum perforatum.

Xanthones are compounds with a diphenyl ether skeleton mainly found in plants and often glycosylated at carbon atoms. Although many C-glycosyltransferases (CGTs) participating in flavone C-glycosylation have been identified, MiCGT from Mangiferaindica, adding sugar to an open-chain benzophenone skeleton, is the only identified xanthone biosynthesis-related CGT. Here, we identified two CGTs from Hypericumperforatum that add sugar to the closed-ring xanthone, but not benzophenone. These CGTs catalyze sugar transfer to the C-4 position of norathyriol (1,3,6,7-tetrahydroxyxanthone) to form isomangiferin (1,3,6,7-tetrahydroxyxanthone 4-C-glucoside), a major xanthone C-glucoside. This is the first study to report CGTs that mediate the direct C-glycosylation of xanthone.

De novo biosynthesis of C-arabinosylated flavones by utilization of indica rice C-glycosyltransferases

Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

Biocatalytic Application of a Membrane‐Bound Coumarin C‐Glucosyltransferase in the Synthesis of Coumarin and Benzofuran C‐Glucosides

AbstractA number of plant C‐glycosyltransferases (CGTs) have been recently identified, whereas the coumarin CGTs have not been found. Here we reported the first identification of a coumarin C‐glucosyltransferase MaCGT fromMorus alba. The whole‐cell biocatalyst harboring MaCGT was exploited and exhibited strictly and efficiently regio‐ and stereo‐specific C‐glucosylation capacity toward structurally different coumarins. The whole‐cell of MaCGT was further applied to chemo‐enzymatically synthesize benzofuran C‐glucosides. Moreover, MaCGT is reported as the first characterized membrane‐bound CGT from plants, although sequence homology search has shown other potentially membrane‐bound CGTs fromM. notabilis; and the presence of the transmembrane domain is critical for its catalytic activity and stability as demonstrated by the C‐glucosylation activity of truncated MaCGT proteins. In addition, the key residues involved in the C‐glucosylation of MaCGT were explored. These findings suggest the diversity of CGTs in the plant kingdom and demonstrate the significant potential of biocatalytic approach to produce structurally diverse C‐glycosides for drug discovery.magnified image

Dissection of the general two-step di-C-glycosylation pathway for the biosynthesis of (iso)schaftosides in higher plants

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

Crystal Structures of the C-Glycosyltransferase UGT708C1 from Buckwheat Provide Insights into the Mechanism of C-Glycosylation.

C-Glycosyltransferases (CGTs) catalyze the formation of C-glycosidic bonds for the biosynthesis of C-glycosides, but the underlying mechanism is unclear. This process improves the solubility and bioavailability of specialized metabolites, which play important roles in plant growth and development and represent rich resources for drug discovery. Here, we performed functional and structural studies of the CGT UGT708C1 from buckwheat (Fagopyrum esculentum). Enzymatic analysis showed that UGT708C1 is capable of utilizing both UDP-galactose and UDP-glucose as sugar donors. Our structural studies of UGT708C1 complexed with UDP-glucose and UDP identified the key roles of Asp382, Gln383, Thr151, and Thr150 in recognizing the sugar moiety of the donor substrate and Phe130, Tyr102, and Phe198 in binding and stabilizing the acceptor. A systematic site-directed mutagenesis study confirmed the important roles of these residues. Further structural analysis combined with molecular dynamics simulations revealed that phloretin binds to the acceptor binding pocket in a bent state with a precise spatial disposition and complementarity. These findings provide insights into a catalytic mechanism for CGTs.

Pathway-specific enzymes from bamboo and crop leaves biosynthesize anti-nociceptive C-glycosylated flavones

C-glycosylated flavones (CGFs) are promising candidates as anti-nociceptive compounds. The leaves of bamboo and related crops in the grass family are a largely unexploited bioresource with a wide array of CGFs. We report here pathway-specific enzymes including C-glycosyltransferases (CGTs) and P450 hydroxylases from cereal crops and bamboo species accumulating abundant CGFs. Mining of CGTs and engineering of P450s that decorate the flavonoid skeleton allowed the production of desired CGFs (with yield of 20–40 mg/L) in an Escherichia coli cell factory. We further explored the antinociceptive activity of major CGFs in mice models and identified isoorientin as the most potent, with both neuroanalgesic and anti-inflammatory effects superior to clinical drugs such as rotundine and aspirin. Our discovery of the pain-alleviating flavonoids elicited from bamboo and crop leaves establishes this previously underutilized source, and sheds light on the pathway and pharmacological mechanisms of the compounds.

Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae

BackgroundC-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H).ResultsIn this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides.ConclusionsThe current study features yeast as a promising host for production of flavone C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation.

Probing and Engineering Key Residues for Bis-C-glycosylation and Promiscuity of a C-Glycosyltransferase

C-Glycosyltransferases (CGTs) are powerful tools for the C-glycosylation of natural and unnatural products. However, CGTs able to catalyze bis-C-glycosylation are very rare and the key amino acids of which have not been uncovered. Here, we discovered a C-glycosyltransferase MiCGTb from Mangifera indica that has the capacity for bis-C-glycosylation. Further studies on active-site motifs revealed that I152 of MiCGTb was the critical amino acid residue for the second C-glycosylation and its S60/V100/T104 residues were pivotal for bis-C-glycosylation activity. Moreover, we developed a panel of variants with acceptor and donor promiscuity by site-directed mutagenesis. Among these variants, a mutant MiCGT-E152L displayed a broader acceptor scope for bis-C-glycosylation, and three mutants of MiCGTb exhibited sugar donor promiscuity toward structurally varied α-d- and β-l-glycosyl donors. Our work provides insights into the pivotal amino acid residues of CGTs for bis-C-glycosylation and biocatalytic tools to effi...

C-Glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants.

Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di-C-glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2-hydroxyflavanones, dihydrochalcone, and their mono-C-glucosides as sugar acceptors and produced corresponding di-C-glucosides. The Km and kcat values of FcCGT toward phloretin were <0.5μm and 12.0sec-1 , and those toward nothofagin (3'-C-glucosylphloretin) were 14.4μm and 5.3sec-1 , respectively; these values are comparable with those of other glycosyltransferases reported to date. Transcripts of both CGT genes were found to concentrate in various plant organs, and particularly in leaves. Our results suggest that di-C-glucosyl flavonoid biosynthesis proceeds via a single enzyme using either 2-hydroxyflavanones or phloretin as a substrate in citrus plants. In addition, Escherichia coli cells expressing CGT genes were found to be capable of producing di-C-glucosyl flavonoids, which is promising for commercial production of these valuable compounds.

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C-glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C-glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C-glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C-glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C-glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P.lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C-glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the invivo function of PlUGT43, the PlUGT43 gene was over-expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.

Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins

The rice C-glycosyltransferase (OsCGT) is one of only a small number of characterised plant C-glycosyltransferases (CGT) known. The enzyme C-glucosylates a 2-hydroxyflavanone substrate with UDP-glucose as the sugar donor to produce C-glucosyl-2-hydroxyflavanones. We tested substrate specificity of the enzyme, using synthetic 2-hydroxyflavanones, and showed it has the potential to generate known natural CGFs that have been isolated from rice and also other plants. In addition, we synthesised novel, unnatural 2-hydroxyflavanone substrates to test the B-ring chemical space of substrate accepted by the OsCGT and demonstrated the OsCGT capacity as a synthetic reagent to generate significant quantities of known and novel CGFs. Many B-ring analogues are tolerated within a confined steric limit. Finally the OsCGT was used to generate novel mono-C-glucosyl-2-hydroxyflavanones as putative biosynthetic intermediates to examine the potential of Desmodium incanum biosynthetic CGTs to produce novel di-C-glycosylflavones, compounds implicated in the allelopathic biological activity of Desmodium against parasitic weeds from the Striga genus.

Enzymatic C-glycosylation: Insights from the study of a complementary pair of plant O- and C-glucosyltransferases

C-Glycosylation presents a rare mode of sugar attachment to the core structure of natural products and is catalyzed by a special type of Leloir C-glycosyltransferases (C-GTs). Elucidation of mechanistic principles for these glycosyltransferases (GTs) is of fundamental interest, and it could also contribute to the development of new biocatalysts for the synthesis of valuable C-glycosides, potentially serving as analogues of the highly hydrolysis-sensitive O‑glycosides. Enzymatic glucosylation of the natural dihydrochalcone phloretin from UDP‑D-glucose was applied as a model reaction in the study of a structurally and functionally homologous pair of plant glucosyltransferases, where the enzyme from rice (Oryza sativa) was specific for C-glycosylation and the enzyme from pear (Pyrus communis) was specific for O-glycosylation. We show that distinct active-site motifs are used by the two enzymes to differentiate between C- and O-glucosylation of the phloretin acceptor. An enzyme design concept is therefore developed where exchange of active-site motifs results in a reversible switch between C/O-glycosyltransferase (C/O-GT) activity. Mechanistic proposal for enzymatic C-glycosylation involves a single nucleophilic displacement at the glucosyl anomeric carbon, proceeding through an oxocarbenium ion-like transition state. Alternatively, the reaction could be described as Friedel&amp;ndash;Crafts-like direct alkylation of the phenolic acceptor.