Structural characterization of Landomycin O and C Glycosyltransferases Zusammenfassung: LanGT2 is the O glycosyltransferase (O GT) that catalyzes the first D olivose attachment on the polyketide aglycone with glycosidic C O bond formation in landomcyin A biosynthesis while UrdGT2 is the C glycosyltransferase (C GT) that catalyzes the formation of a C C bond between the polyketide aglycone and D olivose in urdamycin A biosynthesis. Both proteins are classified as the GT1 family and share a significant 53% protein sequence homology. Swapping only 10 residues of O GT LanGT2 and C GT UrdGT2 and an additional substitution of S8 to A8 creates a mutant protein LanGT2S8Ac a C GT to catalyze the C C bond formation in vivo. Crystal structures of both O GT LanGT2 and C GT LanGT2S8Ac as well as TDP carba D olivose or TDP bound structures have been determined by X ray diffraction to 1.9 2.3 Å resolution. The sugar nucleotide donor binds in the C terminal domain of O GT LanGT2 and C GT LanGT2S8Ac with a similar binding mode in which the carba D olivose is oriented towards the interdomain cleft. Conformational changes of highly flexible helices α8a and α8b are observed during the binding of nucleotide sugar donor and after glycosyltransfer of the D olivose. Helices α8a and α8b are poorly defined in apo structures and become stabilized through the recognition of the carba Dolivose moiety of TDP carba D olivose. After glycosyltransfer of the D olivose moiety in the TDP bound protein helices α8a and α8b become mobile to allow the release of negatively charged thymidine diphosphate. Molecular docking of the aglycone substrate 11 deoxylandomcyinone and tetrangulol in LanGT2 and LanGT2S8Ac illustrated that the aglycone binds in the N terminal domain and provides a mechanistic basis for divergent Oand C glycosylation respectively. The docking models suggest that D137 acts as a catalytic base to abstract a proton from the C8 OH group of aglycone where the orientation of the Oor C nucleophile in relation to the sugar nucleotide is anticipated to distinguish O versus Cglycosylation. These results may provide a blueprint for the rational design of novel GTs with increased substrate promiscuity and or divergent catalytic mechanisms