Binding of 1,n6-ethenoadenosine 5'-triphosphate ATP to an oleandomycin transporter of Streptomyces antibioticus

Abstract

We have previously cloned and characterized an oleandomycin resistance gene coding for the transporter OleB from Streptomyces antibioticus, the producer of the macrolide oleandomycin. We have demonstrated that the first half of this protein (OleB') binds ATP and the substrate oleandomycin. The reagent 1, N6-ethenoadenosine 5'-triphosphate ATP (εATP) is a fluorescent analogue of ATP used to obtain very sensitive information about the structure of a nucleotide binding site. In this work using a technique based on fluorescence quenching of εATP by acrylamide we observed that εATP binds OleB' with a stoichiometry near 1. Data are presented in term of Stern- Volmer and modified Stern-Volmer plots.