title: Structural studies of the spinosyn rhamnosyltransferase, SpnG.
abstract: Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-D-glucose for TDP-D-glucose as the donor substrate is known to result in a >60000-fold reduction in k(cat). Here, we report the structure of SpnG at 1.65 Å resolution, SpnG bound to TDP at 1.86 Å resolution, and SpnG bound to AGL at 1.70 Å resolution. The SpnG-TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG-AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal the features that allow SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Eta A
authors.initials: EA
authors.lastname: Isiorho
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
authors.forename: Adrian T
authors.initials: AT
authors.lastname: Keatinge-Clay
mesh_terms: D002236:Carbohydrate Conformation; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006602:Hexosyltransferases / Q000737:chemistry* / Q000378:metabolism; D018942:Macrolides / Q000737:chemistry* / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000737:chemistry* / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018942:Macrolides; D009702:Nucleoside Diphosphate Sugars; D013942:Thymine Nucleotides; C500915:spinosyn G; C073293:thymidine diphosphate rhamnose; D006602:Hexosyltransferases