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Selenium vs. Sulfur: Investigating the Substrate Specificity of a Selenocysteine Lyase

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Date Issued:
2019
Abstract/Description:
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.
Title: Selenium vs. Sulfur: Investigating the Substrate Specificity of a Selenocysteine Lyase.
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Name(s): Johnstone, Michael, Author
Self, William, Committee Chair
Roy, Herve, Committee Member
Moore, Sean, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2019
Publisher: University of Central Florida
Language(s): English
Abstract/Description: 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.
Identifier: CFE0007659 (IID), ucf:52481 (fedora)
Note(s): 2019-08-01
M.S.
Medicine, Biomedical Sciences
Masters
This record was generated from author submitted information.
Subject(s): selenium -- sulfur -- selenocysteine lyase -- cysteine desulfurase -- NifS -- SclA -- Enterococcus faecalis
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0007659
Restrictions on Access: campus 2020-08-15
Host Institution: UCF

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