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名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成23年3月25日 亀ヶ谷太一氏の博士論文として提出された

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 66-80). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 103-111).

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. / text

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.