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- Title
- IN-FRAME MUTAGENESIS OF GENES ENCODING A SELENIUM-DEPENDENT MOLYBDENUM HYDROXYLASE AND PUTATIVE ACCESSORY PROTEINS IN ENTEROCOCCUS FAECALIS.
- Creator
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Mallard, Christopher, Self, William, University of Central Florida
- Abstract / Description
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Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative selenium-dependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play...
Show moreEnterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative selenium-dependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.
Show less - Date Issued
- 2010
- Identifier
- CFE0003420, ucf:53152
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003420
- Title
- Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis.
- Creator
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Nelson, Samantha, Self, William, Moore, Sean, Rohde, Kyle, University of Central Florida
- Abstract / Description
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A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH),...
Show moreA previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
Show less - Date Issued
- 2014
- Identifier
- CFE0005388, ucf:50455
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005388
- Title
- Selenium vs. Sulfur: Investigating the Substrate Specificity of a Selenocysteine Lyase.
- Creator
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Johnstone, Michael, Self, William, Roy, Herve, Moore, Sean, University of Central Florida
- Abstract / Description
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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...
Show moreSelenium 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.
Show less - Date Issued
- 2019
- Identifier
- CFE0007659, ucf:52481
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007659
