Abstract
The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular inactivation and extracellular reactivation of the antibiotic. Inactivation takes place by transfer of a glucose molecule from a donor (UDP-glucose) to OM, a process catalyzed by an intracellular glucosyltransferase. Glucosyltransferase activity is detectable in cell-free extracts concurrent with biosynthesis of OM. The enzyme has been purified 1,097-fold as a monomer, with a molecular mass of 57.1 kDa by a four-step procedure using three chromatographic columns. The reaction operates via a compulsory-order mechanism. This has been shown by steady-state kinetic studies using either OM or an alternative substrate (rosaramycin) and dead-end inhibitors, and isotopic exchange reactions at equilibrium. OM 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 oleandomycin (GS-OM).
Highlights
The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular inactivation and extracellular reactivation ofthe antibiotic
Inactivation takes place by transfer of a glucose molecule from a donor (OOP-glucose) to OM, a process catalyzed by an intracellular glucosyltransferase
In the case of puromycin, biochemical evidence suggests a role for puromycin N-acetyltransferase in the biosynthesis of the antibiotic [15]
Summary
PURIFICATION AND KINETIC CHARACTERIZATION OF AN OLEANDOMYCIN GLUCOSYLTRANSFERASE*. From the Departamento de Biologia Funcional e Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain. Producer, Streptomyces antibioticus [16] This strain synthesizes an intracellular glucosyltransferase (GTF) enzyme capable of inactivating OM (and a few other macrolides) by glucosylation of the 2' -hydroxyl group of the desosamine present in the antibiotic (Fig. 1). This glucosylated oleandomycin (GS-OM) is the final intracellular product of the pathway. To complete OM biosynthesis, S. antibioticus synthesizes and secretes a second enzyme that has recently been purified [19] This reactivates the antibiotic by catalyzing release of glucose.
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