Browsing by Subject "Polyketide"
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Item In vitro polyketide biocatalysis : triketide building-blocks and enzymology(2013-05) Harper, Andrew David; Keatinge-Clay, Adrian TristanPolyketide products are useful compounds to research and industry but can be difficult to access due to their richness in stereogenic centers. Type I polyketide synthases offer unique engineering opportunities to access natural stereocontrol and resultant complex compounds. The development of a controlled in vitro platform based around type I polyketide synthases is described. It has been used to produce a small library of polyketide fragments on an unprecedented and synthetically-relevant scale and explore polyketide synthase enzymology.Item Inside the microbial weapons factory: structural studies of polyketide biosynthetic machinery(2015-08) Gay, Darren Christian; Keatinge-Clay, Adrian Tristan; Robertus, Jon; Hoffman, Dave; Barrick, Jeff; Ekerdt, JohnPolyketides are a class of small molecules synthesized by a broad spectrum of bacteria, plants, and fungi, and many exhibit powerful bioactive properties. The number of clinically-relevant compounds adapted from polyketide scaffolds is growing, eliciting attempts from synthetic organic chemists to construct polyketide-related compounds in the laboratory from simple chemical building blocks. Unfortunately, the current efficiency by which a skilled artisan can synthesize even small quantities of a polyketide is severely limited by the functional and stereochemical complexity of these compounds. Conceptually, it would be much simpler to genetically reprogram the enzymes responsible for polyketide biosynthesis to produce designer molecules; however, the massive size of polyketide synthase enzymes has hindered efforts towards understanding critical features of their structures and mechanisms. Only very recently has structural information become available for enzymes involved in polyketide biosynthesis, providing an initial glimpse into the inner workings of these subcellular pharmaceutical factories. It will not be possible for mankind to fully realize the potential of engineered polyketide synthases without understanding how their architectures govern the molecules they have evolved to produce. In this work, the structure and mechanism of several enzymes involved in polyketide biosynthesis is investigated. An unprecedented architecture for the ketoreductase-enoylreductase didomain from the second module of the spinosyn polyketide synthase reveals structural divergence from the related mammalian fatty acid synthase, and reconstituted in vitro activity of the enoylreductase domain indicates the isolated enzyme retains activity apart from its parent polyketide synthase module. The dehydratase domain isolated from the tenth module of the rifamycin polyketide synthase, previously hypothesized to only form double bonds with (Z) geometry, was found to have altered stereoselectivity dependent on the carrier handle bound to the substrate. The enoyl-isomerase domain, isolated from the fourteenth module of the bacillaene polyketide synthase, utilizes a catalytic mechanism that relies only on a single active site histidine. A series of ketosynthase domains from trans-acyltransferase polyketide synthases reveal how polyketides bind covalently to the active site of the ketosynthase, and how the flanking subdomain of the ketosynthase is used as an anchor point for the assembly of the polyketide synthase megacomplex.Item Investigation of the post-polyketide synthase (PKS) modifications during spinosyn A biosynthesis in Saccharopolyspora spinosa(2010-08) Kim, Hak Joong; Liu, Hung-wen, 1952-Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis.Item Iridium-catalyzed C-C bond formation : development of crotylation and methallylation reactions through transfer hydrogenation(2012-05) Townsend, Ian A.; Krische, Michael J.; Anslyn, Eric V.Under the conditions of transfer hydrogenation utilizing chromatographically purified ortho-cyclometallated iridium C,O-benzoate precatalysts, enantioselective carbonyl crotylation and methallylation can be performed in the absence of stoichiometric metallic reagents and stoichiometric chiral modifiers. In the case of carbonyl crotylation, use of a preformed precatalyst rather than an in situ generated catalyst results in lower reaction temperatures, providing generally higher diastereoselectivity and yields. By utilizing a more reactive leaving group in chloride over acetate on our methallyl donor, the inherently shorter lifetime of the olefin π-complex is compensated for, giving our group’s first report of reactivity utilizing 1,1-disubstituted allyl donors.Item Structural characterization of post-PKS enzymes involved in spinosyn biosynthesis(2014-05) Isiorho, Eta Amauche; Keatinge-Clay, Adrian Tristan; Liu, Hung-wen, 1952-Saccharopolyspora 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.Item Total syntheses of the neuroregenerative natural products vinaxanthone and xanthofulvin and biosynthetic studies(2012-12) Axelrod, Abram Joseph; Siegel, Dionicio R.; Philip, Magnus D; Michael, Krische J; Keatinge-Clay, Adrian T; Pierce-Shimomura, JonathanTotal syntheses of the neuroregenerative natural products vinaxanthone and xanthofulvin have been accomplished. The synthetic routes to both molecules utilize a highly regioselective furan Diels-Alder cycloaddition - aromatization sequence to furnish the catechol fragment present in both natural products. The pentasubstituted catechol was elaborated to a vinylogous amide which was used twice in both syntheses, exploiting the pseudosymmetry found in vinaxanthone and xanthofulvin. This approach enabled the dimerization of 5,6-dehydropolivione forming vinaxanthone, lending significant evidence to a non-enzymatically driven formation of vinaxanthone in Nature. The total synthesis of vinaxanthone was accomplished in nine steps, the shortest synthesis to date, and an additional route was devised to access a set of analogs for biological study. The first total synthesis of xanthofulvin was accomplished in 18 steps and the convergent nature of the synthetic plan allows for analog synthesis. The sets of vinaxanthone and xanthofulvin analogs will be used to examine their inhibition of Semaphorin3A, a protein which inhibits neuronal regeneration, and is the biological target for both molecules.