Glycosylation of macrolide antibiotics - Purification and kinetic studies of a macrolide glycosyltransferase from Streptomyces antibioticus

The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. Th e glycosyltransferase coded by the oleD gene has been purified 371-fold fro m a Streptomyces lividans clone expressing this protein. The reaction produ ct was isolated, and its structure determined by NMR spectroscopy. The kine tic mechanism of the reaction was analyzed using the macrolide antibiotic l ankamycin (LK) as substrate. The reaction operates via a compulsory order m echanism. This has been shown by steady-state kinetic studies and by isotop ic exchange reactions at equilibrium. LK binds first to the enzyme, followe d by UDP-glucose. A ternary complex is thus formed prior to transfer of glu cose. UDP is then released, followed by the glycosylated lankamycin (GS-LK) . A pH study of the reaction was performed to determine values for the mole cular pK values, suggesting possible amino acid residues involved in the ca talytic process.