UVM Theses and Dissertations
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Print
Author:
Snider, Gregg
Title:
Dept./Program:
Biochemistry
Year:
2013
Degree:
PhD
Abstract:
Selenium (Se) is an essential trace element found in certain biomolecules, most prominently in the 21st coding amino acid selenocysteine (Sec, U), a selenium analog of the sulfur-containing cysteine (Cys, C). Unlike the widely used Cys, Sec is only found in 25 human proteins. This rarity, coupled with its energetically costly mechanism of insertion into proteins, relative to the other 20 commong amino acids, has prompted researchers to postulate Sec confers an important function to human health with the closely related Cys cannot perform. Since most selenoproteins are enzymes, and Cysmutants of WT Sec-enzymes lose significant activity, the pervasive belief in the field is that Sec confers a "catalytic superiority" over Cys in enzymes, due to its unique chemical properties. While Sec does differ from Cys chemically, we and others have demonstrated that WT Cys-orthologs of Sec-enzymes can catalyze the identical enzymatic reaction with near equivalent efficiency, indicating Sec's chemical properties are not required strictly for their mechanistic contributions in enzymes and that additional functions must be served.
This belief is further supported by the fact that Cys has not entirely replaced Sec in the genome. We believe this additional role served by Sec effectively ties Se's unique chemical properties (superior electrophilicity relative to S) to biology, by providing a superior ability to redox cycle, and correspondingly, better protecting Secenzymes from inactivation arising from oxidation, as compared to their Cys-orthologs.
Aim 1 of my dissertation directly tests this hypothesis, employing the mouse mitochondrial thioredoxin reductase (mTR3-GC₁U₂G) as a model selenoenzyme. We first demonstrated the remarkable ability of the full length and truncated mTR3 to resist inactivation from H₂O₂. We also found mTR3[delta]8 effectively able to reduce the Se-oxide containing methaneseleninic acid (CH₃SeO₂⁻), implying that full length mTR3, containing an over-oxidized C-terminal Sec residue, could recover from oxidative inactivation. We also compared the abilities of orthologous WT Sec- and Cys-TR (TR from Drosophila melanogaster-DmTR-SC₁C₂S) enzymes to resist inactivation from a variety of oxidants. This comparison was further enhanced through the generation of a DmTR "Sec-rescue" mutant (DmTR-SC₁U₂G). We demonstrated both Sec-TRs to retain significantly more activity following oxidant exposure then the WT Cys-TR, proving the superior ability of Sec to redox cycle and protect enzymes from oxidant-induced inactivation over Cys. This relationship aslo held true with analogous Cys- and Sec-TR constructs from Plasmodium falciparum.
Aim II of my dissertation is comprised of a mechanistic investigation into the unique enzymology displayed by the type II C-terminal redox center of P. falciparum TR, which contains four bridging amino acids between each catalytic Cys residue (PfTR-C₁GGGKC₂G). Our results demonstrate PfTR is heavily reliant on the native size, geometry, dynamics, and length of the type II 20-membered disulfide-containing ring activity loss with Trx as substrate. We also demonstrated a chimeric type Ib PfTR-C₁C₂G mutant failed to reduce Trx, but a chimeric type Ia PfTR-C₁C₂G Sec-mutant displayed robust Trx reductase activity. Aim II experiments also explored the substrate specificity of PfTr, relative to type I TRs, and our results demonstrate the type II PfTR to reduce a broad spectrum of substrates similar to the type I high Mr TRs.
This belief is further supported by the fact that Cys has not entirely replaced Sec in the genome. We believe this additional role served by Sec effectively ties Se's unique chemical properties (superior electrophilicity relative to S) to biology, by providing a superior ability to redox cycle, and correspondingly, better protecting Secenzymes from inactivation arising from oxidation, as compared to their Cys-orthologs.
Aim 1 of my dissertation directly tests this hypothesis, employing the mouse mitochondrial thioredoxin reductase (mTR3-GC₁U₂G) as a model selenoenzyme. We first demonstrated the remarkable ability of the full length and truncated mTR3 to resist inactivation from H₂O₂. We also found mTR3[delta]8 effectively able to reduce the Se-oxide containing methaneseleninic acid (CH₃SeO₂⁻), implying that full length mTR3, containing an over-oxidized C-terminal Sec residue, could recover from oxidative inactivation. We also compared the abilities of orthologous WT Sec- and Cys-TR (TR from Drosophila melanogaster-DmTR-SC₁C₂S) enzymes to resist inactivation from a variety of oxidants. This comparison was further enhanced through the generation of a DmTR "Sec-rescue" mutant (DmTR-SC₁U₂G). We demonstrated both Sec-TRs to retain significantly more activity following oxidant exposure then the WT Cys-TR, proving the superior ability of Sec to redox cycle and protect enzymes from oxidant-induced inactivation over Cys. This relationship aslo held true with analogous Cys- and Sec-TR constructs from Plasmodium falciparum.
Aim II of my dissertation is comprised of a mechanistic investigation into the unique enzymology displayed by the type II C-terminal redox center of P. falciparum TR, which contains four bridging amino acids between each catalytic Cys residue (PfTR-C₁GGGKC₂G). Our results demonstrate PfTR is heavily reliant on the native size, geometry, dynamics, and length of the type II 20-membered disulfide-containing ring activity loss with Trx as substrate. We also demonstrated a chimeric type Ib PfTR-C₁C₂G mutant failed to reduce Trx, but a chimeric type Ia PfTR-C₁C₂G Sec-mutant displayed robust Trx reductase activity. Aim II experiments also explored the substrate specificity of PfTr, relative to type I TRs, and our results demonstrate the type II PfTR to reduce a broad spectrum of substrates similar to the type I high Mr TRs.