Functional analysis of oleY L-oleandrosyl 3-O-methyltransferase of the oleandomycin biosynthetic pathway in Streptomyces antibioticus

Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine . oleY codes for a methyltransferase involved in the biosynthesis of L-olea ndrose. This gene was overexpressed in Escherichia coli to form inclusion b odies and in Streptomyces lividans, producing a soluble protein. S. lividan s overexpressing oleY was used as a biotransformation host, and it converte d the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivati ve, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives w ere also substrates for the OleY methyltransferase: L-rhamnosyl- and L-myca rosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kD a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chro matography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by t he presence of several divalent cations, being maximal with Co2+. The S. an tibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to t he oleandolide aglycon, which is then converted into L-oleandrose by the Ol eY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis , in which L-oleandrose is transferred to the aglycon by a glycosyltransfer ase.