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. 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.
Quirós
L M
LM
Departamento de Biología Funcional, Universidad de Oviedo, 33006 Oviedo, Spain.
Carbajo
R J
RJ
Braña
A F
AF
Salas
J A
JA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002384:Catalysis; D004917:Erythromycin / Q000031:analogs & derivatives / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009827:Oleandomycin / Q000378:metabolism; D017510:Protein Folding; D013303:Streptomyces antibioticus / Q000201:enzymology*; D014530:Uridine Diphosphate / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D000900:Anti-Bacterial Agents; D014530:Uridine Diphosphate; D004917:Erythromycin; C100149:kujimycin B; D005964:Glucosyltransferases; C075034:macrolide glycosyltransferase; D009827:Oleandomycin; D014532:Uridine Diphosphate Glucose