title: In vitro reconstitution of EryCIII activity for the preparation of unnatural macrolides.
abstract: EryCIII is a desosaminyltransferase that converts an inactive macrolide precursor to a biologically active antibiotic. It may have potential for the synthesis of unnatural macrolides with useful biological activities. However, it has been difficult to reconstitute the activity of EryCIII in vitro. We report here that purified, inactive EryCIII can be converted to an active catalyst by the addition of another protein encoded in the same gene cluster, EryCII. The EryCII-treated protein retains activity even when EryCII is removed. We also show that AknT, an activator protein from an unrelated gene cluster, is capable of activating EryCIII. Although the mechanism of activation is not yet understood, we have concluded from these experiments that these antibiotic Gtf activator proteins do not function to deliver substrates to EryCIII and do not exert their effects by forming stable complexes with the Gtf during the glycosyltransfer reaction. We report that activated EryCIII is capable of utilizing an alternative sugar donor, so these results lay the groundwork for the production of novel macrolides.
authors.affiliation: Department of Microbiology and Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Yanqiu
authors.initials: Y
authors.lastname: Yuan
authors.forename: Hak Suk
authors.initials: HS
authors.lastname: Chung
authors.forename: Catherine
authors.initials: C
authors.lastname: Leimkuhler
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: Daniel
authors.initials: D
authors.lastname: Kahne
authors.forename: Suzanne
authors.initials: S
authors.lastname: Walker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D004789:Enzyme Activation; D016695:Glycosyltransferases / Q000737:chemistry*; D066298:In Vitro Techniques; D018942:Macrolides / Q000138:chemical synthesis* / Q000737:chemistry; D008968:Molecular Conformation; D016930:Saccharopolyspora / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D018942:Macrolides; D016695:Glycosyltransferases; C111821:EryCIII protein, Saccharopolyspora erythraea