Honors College Thesis
 

Partial Characterization of the Danio rerio and Xenopus tropicalis MT-­‐Ox Enzyme Involved in Gadusol Biosynthesis

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  • Ultraviolet (UV)-protective compounds, such as mycosporine-like amino acids (MAA) are critical for the survival of marine organisms exposed to high-solar radiation. A chemically similar compound, gadusol, was discovered in eggs of the atlantic cod in 1980, and was originally thought to be derived from MAAs that had been ingested. However, a two-gene cluster was found in Danio rerio that was recently confirmed to encode gadusol biosynthesis is also found in the genome of the atlantic cod and other fish, amphibians, and reptiles. One gene encodes a sugar phosphate cyclase, 2-epi-5epi-valione synthase (EEVS) and a previously uncharacterized gene which appears by sequence comparisons to encode a protein designated “MT-Ox” that has a methyltransferase domain connected to an oxidoreductase domain. While there have been several studies on EEVS enzymes, MT-Ox is not as well characterized. Here, I report on the development of a recombinant E. coli expression system of Xenopus tropicalis MT-Ox protein. The recombinant Xenopus tropicalis protein along with the Danio rerio MT-Ox protein were used for characterization and crystallization. Both proteins expressed well, were purified using cobalt affinity chromatography, and then studied by limited proteolysis using the enzymes chymotrypsin or trypsin. Some success was had at obtaining a fragment that I hypothesize is the “Ox” domain, which can now be tested using protein crystallization trials and analysis. Key Words: gadusol, methyltrasferase-­‐oxidoreductase, limited proteolysis, Xenopus tropicalis
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