RESUMEN
Anthracyclines are an important class of natural antitumor drugs. They have a conservative aromatic tetracycline backbone that is substituted with different deoxyglucoses. The deoxyglucoses are crucial for the biological activity of many bacterial natural products after the proper modification from glycosyltransferases (GTs). The difficulty in obtaining highly purified active GTs has prevented biochemical studies on natural product GTs. In this paper, a new Escherichia coli fusion plasmid pGro7', which introduces the Streptomyces coelicolor chaperone genes groEL1, groES and groEL2, was constructed. The glycosyltransferase DnmS from Streptomyces peucetius ATCC 27952 was co-expressed with the plasmid pGro7', and unprecedented high-efficiency and soluble expression of DnmS in the E. coli expression system was realized. Subsequently, the reverse glycosylation reaction characteristics of DnmS and DnmQ were verified. We found that DnmS and DnmQ had the highest enzyme activity when they participated in the reaction at the same time. These studies provide a strategy for the soluble expression of GTs in Streptomyces and confirm the reversibility of the catalytic reaction of GTs. This provides a powerful method for the production of active anthracyclines and to enhance the diversity of natural products.
RESUMEN
Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose-phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose-phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP-glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP · GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or ß-glucose or a mixture of both anomers can account for the observed electron density of the UDP-glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.