University thesis:
Dissertation, Albert-Ludwigs-Universität Freiburg, 2013
Footnote:
Description:
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