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dc.contributor Miller-Schulze, Justin en_US
dc.contributor McCarthy Hintz, Mary en_US
dc.contributor.advisor Savage, Thomas J. en_US
dc.contributor.author Badarou, Adriana
dc.date.accessioned 2021-05-17T16:32:44Z
dc.date.available 2021-05-17T16:32:44Z
dc.date.issued 2021-05-17
dc.date.submitted 2020-08
dc.identifier.uri http://hdl.handle.net/10211.3/218998
dc.description Thesis (M.S., Chemistry (Biochemistry))--California State University, Sacramento, 2020. en_US
dc.description.abstract Pseudo-nitzchia diatoms are a genus of microorganisms that live along the coast of California, Canada, Europe, Australia, Central and South America. These organisms produce a neurotoxin known as domoic acid (DA), which is responsible for causing amnesic shellfish poisoning in marine animals. The biological pathway and function of DA is still unclear. However, recent research has indicated N-geranyl-L-glutamic acid is a precursor to DA. N-geranyl-L-glutamic acid is derived from two metabolites: glutamate and geranyl diphosphate. Geranyl diphosphate is generated from isopentenyl diphosphate and dimethylallyl diphosphate in a reaction catalyzed by a member of the prenyltransferase family: geranyl diphosphate synthase. The goal of this project is to identify the gene that encodes the enzyme that generates the geranyl diphosphate precursor to DA in Pseudo-nitzschia multiseries. The genome of P. multiseries contains seven expressed genes that encode prenyltransferase-like proteins, although it is unclear which of these generates geranyl diphosphate for DA production. Here we focus on identifying the product specificity of the prenyltransferase transcripts Psemu1 and Psemu2. Amino acid sequence homology searches were performed using the bioinformatic tool Basic Local Alignment Search Tool (BLAST) to identify the most homologous proteins which have been previously characterized experimentally to validate their product specificity: geranyl diphosphate, farnesyl diphosphate or geranylgeranyl diphosphate. Additionally, using Iterative Threading ASSEmbly Refinement (I-TASSER), three dimensional structure models were generated of Psemu1 and Psemu2 protein to analyze the catalytic pocket and identify residues in close proximity to the First Aspartate-Rich Motif (FARM) and the Second Aspartate-Rich Motif (SARM) regions. Expression vectors for Psemu1 and Psemu2 proteins were then generated using synthetic DNA technology. Expressed protein was then generated and isolated using Ni2+ agarose affinity chromatography, then assayed for catalytic activity by LC-MS/MS. Significant amounts of insoluble protein aggregates accumulated and little soluble protein was isolated. Furthermore, geranyl diphosphate or any larger isoprenyl diphosphate chains were not detected in prenyltransferase assays of the soluble protein that did accumulate. In conclusion, no catalytic activity was detected by LC-MS/MS suggesting that prenyltransferase-like transcripts Psemu1 and Psemu2 in P. multiseries may not contribute to the formation of geranyl diphosphate. en_US
dc.description.sponsorship Chemistry en_US
dc.language.iso en_US en_US
dc.subject Domoic acid en_US
dc.subject Pseudo-nitzchia en_US
dc.subject Geranyl diphosphate en_US
dc.title Identifying the enzyme that catalyzes the synthesis of geranyl diphosphate, a precursor to domoic Acid in Pseudo-nitzchia en_US
dc.type Thesis en_US

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