Abstract
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. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.
Highlights
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
Most macrolides are produced by streptomycetes and the biosynthesis of a potentially lethal antibiotic by these microorganisms requires the existence of a self-resistance mechanism in the producing microorganism to avoid cell suicide
We report the characterization of the macrolide glycosyltransferase (MGT) coded by the oleD gene and the elucidation of the structure of the glucosylated oleandomycin (GS-OM)
Summary
PURIFICATION AND KINETIC STUDIES OF A MACROLIDE GLYCOSYLTRANSFERASE FROM STREPTOMYCES ANTIBIOTICUS*. Macrolides constitute a group of antibiotics mainly active against Gram-positive bacteria and having clinical application in the treatment of bacterial infections In their structures one or several saccharidic moieties are present, attached to a macrocyclic lactone. One of the resistance mechanisms so far described in macrolide producing strains is the existence of intracellular glycosyltransferases These enzymes inactivate macrolides by glycosylation of a hydroxyl group present in one of the 6-deoxyhexoses attached to the macrolactone ring. In the same microorganism, a gene codifying a macrolide glycosyltransferase (MGT) activity able to transfer a glucose moiety to many macrolides and located outside the oleandomycin biosynthesis gene cluster (OleD) has been cloned (7). Kinetic studies to obtain information about the enzyme mechanism are performed, as well as pH studies to determine values for the molecular pK values in order to suggest possible amino acid residues involved in the catalytic process
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