Abstract
Domoic acid is a potent neurotoxin that can cause amnesic shellfish poisoning. It causes irreversible lesions in the brain, heart, and other essential organs and is produced in the marine diatom Pseudo-nitzschia. The exact mechanism for the production of domoic acid is unclear, but it has been determined that it is formed from geranyl diphosphate and an activated glutamate. The production of geranyl diphosphate is catalyzed by short-chain prenyltransferases which catalyze condensation reactions between isopentenyl diphosphate and dimethylallyl diphosphate. The genome of Pseudo-nitzschia multiseries has been shown to have seven different genes that might encode short-chain prenyltransferases. The goal of this study is to determine whether one of these homologous genes encodes a prenyltransferase that generates geranyl diphosphate. A cDNA was amplified and cloned into an expression vector, and the expressed enzyme was purified by Ni2+-agarose affinity chromatography to isolate protein suitable for use in catalytic activity assays. The assay products were analyzed by LC-MS to determine whether the prenyltransferase encoded by this particular gene produced geranyl diphosphate or a longer allylic diphosphate. LC-MS analysis did not detect production of any allylic diphosphate, with the conclusion that due to various factors this particular gene does not contribute to the production of geranyl diphosphate.