Selenium is a vital micronutrient in many organisms. While traces are required for survival, excess amounts are toxic; thus, selenium can be regarded as a biological "double-edged sword". Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. In this work, we add knowledge to the mechanism for selenium-over-sulfur specificity in hopes of further understanding the controlled regulation of selenium trafficking and the prevention of its toxicity. We have identified SclA, a selenocysteine lyase in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for L-selenocysteine over L-cysteine. Human selenocysteine lyase contains a residue, D146, which plays a significant role in determining its specificity. A D146K mutation eliminated this trait, allowing non-specific L-cysteine degradation. Using computational biology, we identified an orthologous residue in SclA, H100, and generated mutant enzymes with site-directed mutagenesis. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying levels of activity towards L-selenocysteine, hinting at a catalytic role for H100. Additionally, L-cysteine acted as a competitive inhibitor towards all enzymes with higher affinity than L-selenocysteine. Finally, our experiments revealed that SclA possessed extremely poor cysteine desulfurase activity with each mutation exhibiting subtle changes in turnover. Our findings offer key insight into the molecular mechanisms behind selenium-over-sulfur specificity and may further elucidate the role of selenocysteine lyases in vivo.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-7511 |
| Date | 01 January 2019 |
| Creators | Johnstone, Michael |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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