Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.

On the Catalytic Activity of a GT1 Family Glycosyltransferase from Streptomyces venezuelae ISP5230.

GT1 family glycosyltansferase, Sv0189, from Streptomyces venezuelae ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars. In addition to polyphenols, including anthraquinones, simple aromatics containing primary or secondary alcohols, a variety of complex natural products and synthetic drugs were glucosylated or xylosylated by Sv0189. Regioselectivity was established through the isolation and characterization of glucosylated products. Sv0189 and homologous proteins are widely distributed among Streptomyces species, and their apparent substrate promiscuity reveals potential for their development as biocatalysts for glycodiversification.

Enzyme-catalyzed synthesis of heptyl-ß-glycosides: effect of water coalescence at high temperature.

Alkyl glycosides can be synthesized by glycosidases in organic media with limited amounts of water. These systems, however, limit the solubility of the sugar substrates and decrease reaction yields. Herein we report the enzymatic synthesis of heptyl-ß-glycosides in heptanol catalyzed by a hyperthermophilic ß-glycosidase at 90°C. Our results indicate that dispersion of water in heptanol changes with time producing coalescence of water at the bottom of the reactor, playing a key role in the reaction yield. Water-soluble substrate, enzyme and products are concentrated in the aqueous phase, according to their partition coefficients, promoting side reactions that inactivate the enzyme. Reaction yield of heptyl-ß-glycosides was 35% relative to lactose, at 7% water. The increase in the water phase to 12% diminished the enzyme inactivation and increased the heptyl-ß-glycosides yield to 52%. Surface-active compounds, SDS and octyl glucoside, increased water dispersion but were unable to prevent coalescence.

Synthetic tools for the characterization of galactofuranosyl transferases: glycosylations via acylated glycosyl iodides.

With the aim of developing synthetic tools for the characterization of galactofuranosyltransferases, the synthesis of 9-decenyl glycosides of D-Manp, D-Galf, and ß-D-Galf-(1→3)-D-Manp was targeted. The interest in the alkenyl aglycone arises via potential conjugation reactions, once the terminal double bond has been conveniently functionalized. The glycosylation of ß-D-Galf-(1→3)-D-Manp was attempted by two different approaches: the trichloroacetimidate method and the glycosylation via the glycosyl iodide. The conditions for the latter were established on the basis of glycosylation assays of per-O-acetylmannose. On the other hand, the study of glycosylation reactions via per-O-benzoylated galactofuranosyl iodide confirms the versatility of glycosyl iodides as donors.