The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery.

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The catalytic promiscuity of a new glycosyltransferase (UGT73AE1) from Carthamus tinctorius was explored. UGT73AE1 showed the capability to glucosylate a total of 19 structurally diverse types of acceptors and to generate O-, S-, and N-glycosides, making it the first reported trifunctional plant glycosyltransferase. The catalytic reversibility and regioselectivity were observed and modeled in a one-pot reaction transferring a glucose moiety from icariin to emodin. These findings demonstrate the potential versatility of UGT73AE1 in the glycosylation of bioactive natural products.

c Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ϳ26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.A grobacterium tumefaciens is well known for its ability to induce crown gall tumors in dicotyledonous plants and mediate interkingdom genetic transfer, for that it is widely used in plant molecular biology and biotechnology (1). Interestingly, some strains of this species are also able to degrade xenobiotics such as cyanuric acid, iminodisuccinate, and methylene urea (2-4). We isolated A. tumefaciens strain S33, which has the strong ability to degrade the natural alkaloid nicotine from the rhizospheric soil of a tobacco plant (5, 6).Nicotine is a major alkaloid in tobacco, which causes tobacco addiction and may result in diseases such as pulmonary disease and cancer (7,8), and it is the primary toxic compound in tobacco wastes. The tobacco-manufacturing process and all activities using tobacco produce a large amount of solid or liquid waste containing high concentrations of nicotine, which are classified as "toxic and hazardous wastes" by the European Union (9). Therefore, detoxification of tobacco wastes is a major concern for public health and the environment. The discovery of nicotine degradation by microorganisms provides an alternative way to dispose of such wastes (10-12).Microbial degradation of nicotine attracts attention because it represents a method to treat the tobacco waste...

Background: Plant flavonoid prenyltransferases (FPTs) transfer prenyl moiety to flavonoid cores and have previously been identified only in Leguminosae. Results: The newly identified moraceous FPTs, MaIDT, and CtIDT, are distantly related to leguminous FPTs and feature catalytic regioselectivity and promiscuity. Conclusion: MaIDT and CtIDT evolved independently from leguminous FPTs. Significance: These findings are valuable for identifying additional evolutionarily different non-Leguminosae FPTs.

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-Dand β-Lglycosyl donors. Our work provides insights into the pivotal amino acid residues of CGTs for bis-C-glycosylation and biocatalytic tools to efficiently produce structurally diverse bis-C-glycosides with two identical or different sugar moieties in drug discovery.

A green and cost-effective process for the convenient synthesis of acylphloroglucinol 3-C-glucosides from 2-O-glucosides was exploited using a novel C-glycosyltransferase (MiCGTb) from Mangifera indica. Compared with previously characterized CGTs, MiCGTb exhibited unique de-O-glucosylation promiscuity and high regioselectivity toward structurally diverse 2-O-glucosides of acylphloroglucinol and achieved high yields of C-glucosides even with a catalytic amount of uridine 5'-diphosphate (UDP). These findings demonstrate for the first time the significant potential of a single-enzyme approach to the synthesis of bioactive C-glucosides from both natural and unnatural acylphloroglucinol 2-O-glucosides.