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dc.contributor.advisorKeatinge-Clay, Adrian Tristan
dc.contributor.advisorLiu, Hung-wen, 1952-
dc.creatorIsiorho, Eta Amaucheen
dc.date.accessioned2015-04-07T14:09:28Zen
dc.date.accessioned2018-01-22T22:27:37Z
dc.date.available2018-01-22T22:27:37Z
dc.date.issued2014-05en
dc.date.submittedMay 2014en
dc.identifier.urihttp://hdl.handle.net/2152/29246en
dc.descriptiontexten
dc.description.abstractSaccharopolyspora spinosa is a rare actinomycete that synthesizes the secondary metabolite spinosyn A, which is an active ingredient in several important commercial insecticides. Spinosyn aglycone formation occurs via a type I polyketide synthase. After release of the polyketide chain from the synthase, various tailoring enzymes modify the aglycone core. These unique enzyme transformations result in unusual structural characteristics found in spinosyn A. The enzymes SpnG, SpnP, SpnF and SpnL each perform a key reaction during post-PKS processing. The work presented in this dissertation focuses on the structural determination and analysis of SpnG, SpnP, SpnF and SpnL. SpnG, which naturally catalyzes the 9-OH rhamnosylation of spinosyn, is capable of adding diverse sugars to the spinosyn aglycone from TDP-hexoses, such as TDP-glucose. However, the substitution of UDP-glucose for TDP-glucose as the donor substrate is known to result in a >60,000-fold reduction in k [subscript cat]. The structure of SpnG at 1.65 Å resolution, the 1.86 Å resolution structure of SpnG bound to TDP, and the 1.70 Å resolution structure of SpnG bound to AGL were determined. The SpnG-TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. The SpnG-AGL complex shows that SpnG binds the acceptor substrate primarily through hydrophobic interactions and implicates H13 as the potential catalytic base. A model for how rhamnose binds in the active site was constructed to elucidate which features enable SpnG to transfer diverse hexoses. SpnP transfers forosamine from a TDP-D-forosamine donor substrate to a spinosyn pseudoaglycone acceptor substrate. The structures of SpnP and its complex with TDP were determined to 2.50 Å and 3.15 Å resolution, respectively. SpnP possesses a structural feature that has only been previously observed in a related glycosyltransferase, which employs an auxiliary protein that aids in its catalysis. This unique feature may be a used as a predictive motif of glycosyltransferases that interact with an auxiliary protein. SpnF and SpnL are two novel S-adenosyl-L-methionine dependent cyclases. Structural data was utilized in order to gain insight into the unusual cycloaddition catalyzed by the putative Diels-Alderase and Rauhut-Currierase, SpnF and SpnL, respectively. Together these structures provide valuable insights into the unusual mechanisms involved in spinosyn biosynthesis.en
dc.format.mimetypeapplication/pdfen
dc.language.isoenen
dc.subjectBiosynthesisen
dc.subjectSpinosynen
dc.subjectStructuralen
dc.subjectX-rayen
dc.subjectPolyketideen
dc.subjectCyclaseen
dc.subjectGlycosyltransferaseen
dc.titleStructural characterization of post-PKS enzymes involved in spinosyn biosynthesisen
dc.typeThesisen
dc.description.departmentBiochemistryen
dc.date.updated2015-04-07T14:09:28Zen


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