User:Emma Ste.Marie/Sandbox 1: Difference between revisions

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'''Motifs and Domains''':
'''Motifs and Domains''':
Several Pfam domains (described by The European Bioinformatics Institute (EMBL-EBI)) present in mTrxR1 include: a glutaredoxin domain, pyridine nucleotide-disulphide oxidoreductase domain, pyridine nucleotide-disulphide oxidoreductase dimerisation domain, and nucleotide phosphate-binding region.
Several Pfam domains (described by The European Bioinformatics Institute (EMBL-EBI)) present in mTrxR1 include: a glutaredoxin domain, pyridine nucleotide-disulphide oxidoreductase domain, pyridine nucleotide-disulphide oxidoreductase dimerisation domain, and nucleotide phosphate-binding region.
Link to Pfam information on mTrxR1: [http://pfam.xfam.org/family/PF00462.23] [http://pfam.xfam.org/structure/3qfa]
Link to Pfam information on mTrxR1: [http://pfam.xfam.org/family/PF00462.23] [http://pfam.xfam.org/structure/3qfa]
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'''Interaction of mTrxR with Trx:'''
'''Interaction of mTrxR with Trx:'''
The first crystal structure of Type 1 mTrxR in complex with its Trx substrate was solved by Karin Fritz-Wolf, et al. in 2011 and gave much information into structural details of the enzyme. Their crystal structure confirms that mTrxR1 possesses two redox centres, both of which are required for catalysis. The N-terminally located redox active disulfide (Cys59-Cys64) is buried in the protein, whereas the second, C-terminally located redox active selenosulfide (Cys497-Sec498) is positioned on a flexible, highly accessible C-terminal tail of the other subunit[5]. Complex formation of mTrxR1 with Trx involves the generation of an intermolecular disulphide bond between the catalytic residues Cys32 of Trx and Sec498 of mTrxR1, which is stabilized by several other interactions. Some of the other interactions include: Trp resudies buried with their indole rings at the interface region within van der Waals distance (hydrophobic interactions), hydrogen bonds between amino acid side chain residues (which forms salt bridges), and other interactions involving several oppositely charged residues. All residues involved in forming the intermediate are highly conserved in mammals[5]. It is important to note in this crystal structure that penultimate Sec residues were mutated to Cys residues (''E. coli'', the expression vector used, does not have the machinery to incorporate Sec into proteins). Additionally, the resolving Cys residue was mutated to alanine to keep Trx bound.
The first crystal structure of Type 1 mTrxR in complex with its Trx substrate was solved by Karin Fritz-Wolf, et al. in 2011 and gave much information into structural details of the enzyme. The N-terminally located redox active disulfide (Cys59-Cys64) is buried in the protein, whereas the second, C-terminally located redox active selenosulfide (Cys497-Sec498) is positioned on a flexible, highly accessible C-terminal tail of the other subunit[5]. Complex formation of mTrxR1 with Trx involves the generation of an intermolecular disulphide bond between the catalytic residues Cys32 of Trx and Sec498 of mTrxR1, which is stabilized by several other interactions. Some of the other interactions include: hydrophobic packing (mainly Trp resudies buried with their indole rings at the interface region), hydrogen bonds between amino acid side chain residues, and other interactions involving several oppositely charged residues (electrostatic interactions). All residues involved in forming the intermediate are highly conserved in mammals[5]. It is important to note in this crystal structure that penultimate Sec residues (one on each monomer) were mutated to Cys residues (''E. coli'', the expression vector used, does not have the machinery to incorporate Sec into proteins). Additionally, the resolving Cys residue was mutated to alanine to keep Trx bound.


[[Image:Ctermredoxsmall.png|1000px|right|thumb|PDB: 3QFA with features highlighted. C-terminal redox centre of mTrxR: green ribbons are one monomer of mTrxR1, and blue ribbons are the other monomer. In this crystal structure, Trx (yellow ribbons) is bound to mTrxR1, and this disulfide linkage is highlighted by yellow and green spheres. It is important to note that Sec498 was mutated to Cys498, and this Cys498 residue is shown forming a disulfide with Trx. Shown in green stick is another mutation: in this crystal structure, the resolving Cys residue was mutated to Alanine to ensure Trx stays bound to mTrxR1.]]
[[Image:Ctermredoxsmall.png|1000px|right|thumb|PDB: 3QFA with features highlighted. C-terminal redox centre of mTrxR: green ribbons are one monomer of mTrxR1, and blue ribbons are the other monomer. In this crystal structure, Trx (yellow ribbons) is bound to mTrxR1, and this disulfide linkage is highlighted by yellow and green spheres. It is important to note that Sec498 was mutated to Cys498, and this Cys498 residue is shown forming a disulfide with Trx. Shown in green stick is another mutation: in this crystal structure, the resolving Cys residue was mutated to Alanine to ensure Trx stays bound to mTrxR1.]]
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'''Role of Selenocysteine in Type 1 mTrxRs:'''
'''Role of Selenocysteine in Type 1 mTrxRs:'''
Selenocysteine (Sec, U) is the 21st amino acid, and is structurally and chemically similar to cysteine (Cys, C). Unlike the other 20 amino acids, inertion of Sec into a protein is very complicated because a UGA stop codon must be recoded as a sense codon for Sec. Mammalians posses special Sec-insertion machinery required for this process, such as a special stem-loop structure called a selenocysteine insertion sequence element in the 3’ untranslated region of the mRNA, special SECIS binding proteins, and selenocysteine elongation factors that bind specialized tRNA. The complexity of the process of inserting Sec into a protein signifies that Sec must fulfil a chemical function that conventional Cys residues cannot.  
Selenocysteine (Sec, U) is the 21st amino acid, and is structurally and chemically similar to cysteine (Cys, C). Unlike the other 20 amino acids, insertion of Sec into a protein is very complicated because a UGA stop codon must be recoded as a sense codon. Higher level eukaryotes posses special Sec-insertion machinery required for this process, such as a special stem-loop structure called a selenocysteine insertion sequence element (SECIS) in the 3’ untranslated region of the mRNA, special SECIS binding proteins, and selenocysteine elongation factors that bind specialized tRNA. The complexity of the process of inserting Sec into a protein signifies that Sec must fulfill a chemical function that conventional Cys residues cannot.  
 
The role of Sec in mTrxR is not fully understood. One popular hypothesis is that Sec accelerates thiol disulfide exchange reactions in enzymes, thus conferring a kinetic advantage to Sec enzymes. Sec can accelerate thiol disulfide exchange reactions in three different ways [2, 6]. First, Sec can enhance the rate of the enzyme by being a better nucleophile than Cys (Figure 2A). This is a reasonable hypothesis considering the lower pKa of Sec (pKa ~ 5.5) compared to Cys (pKa ~ 8.0); Sec would be deprotonated at a much lower pH (especially at physiological pH), making it a reactive nucleophile [2, 6]. Second, Sec is a better leaving group than Cys, and this accelerates the exchange reaction (Figure 2C). Because the acidity of a selenol is ~1000 times greater than a thiol, selenols are superior leaving groups[2, 6]. Third, Sec is a better electrophile than Cys, and this greater electrophilicity accelerates the reaction (Figure 2B). The greater tolerance for hypervalency of selenium has an important consequence, that nucleophilic attack on selenium (which typically forms or passes through hypervalent intermediates) usually occurs much more rapidly than at sulfur, since the intermediate selenium compounds are lower in energy than sulfur analogs[2, 6]. The Hondal research group has provided much evidence supporting the idea that mTrxR1 uses Sec as a superior electrophile[2, 7].  
The role of Sec in mTrxR is not fully understood. One popular hypothesis is that Sec accelerates thiol disulfide exchange reactions in enzymes, thus conferring a kinetic advantage to Sec enzymes. Sec can accelerate thiol disulfide exchange reactions in three different ways [2, 6]. First, Sec can enhance the rate of the enzyme by being a better nucleophile than Cys (Figure 2A). This is a reasonable hypothesis considering the lower pKa of Sec (pKa ~ 5.5) compared to Cys (pKa ~ 8.0); Sec would be deprotonated at a much lower pH (especially at physiological pH), making it a reactive nucleophile [2, 6]. Second, Sec is a better leaving group than Cys, and this accelerates the exchange reaction (Figure 2C). Because the acidity of a selenol is ~1000 times greater than a thiol, selenols are superior leaving groups[2, 6]. Third, Sec is a better electrophile than Cys, and this greater electrophilicity accelerates the reaction (Figure 2B). The greater tolerance for hypervalency of selenium has an important consequence, that nucleophilic attack on selenium (which typically forms or passes through hypervalent intermediates) usually occurs much more rapidly than at sulfur, since the intermediate selenium compounds are lower in energy than sulfur analogs[2, 6]. The Hondal research group has provided much evidence supporting the idea that mTrxR1 uses Sec as a superior electrophile[2, 7].  


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Another hypothesis for the role of Sec in mTrxR (and other seleno-enzymes) is that the Sec residue confers resistance to inactivation by oxidation to the enzyme. In 2013, G. Snider ''et al''. showed that mTrxR is able to resist inactivation by oxidation from a variety of oxidants including hydrogen peroxide, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypocyanous acid. A Cys-orthologue was also exposed to the oxidants, and they Cys- and Sec-enzymes were directly compared [8]. It would found that Sec-mTrxR is far superior to the Cys-orthologue TrxR in resisting inactivation by oxidation. The greatest divergence between selenium and sulfur chemistry
Another hypothesis for the role of Sec in mTrxR (and other seleno-enzymes) is that the Sec residue confers resistance to inactivation by oxidation to the enzyme. In 2013, G. Snider ''et al''. showed that mTrxR1 is able to resist inactivation by oxidation from a variety of oxidants including hydrogen peroxide, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypocyanous acid. A Cys-orthologue was also exposed to the oxidants, and the Cys- and Sec-enzymes were directly compared [8]. It was found that Sec-mTrxR is far superior to the Cys-orthologue TrxR in resisting inactivation by oxidation. The greatest divergence between selenium and sulfur chemistry
occurs in the redox reactions of the two elements. Selenium is both a good nucleophile and a good electrophile, and this property allows it to easily cycle between reduced and oxidized states without becoming permanently oxidized [2,8]. These observations lead to the hypothesis that Sec confers a redox advantage to enzymes instead of a kinetic advantage.  
occurs in the redox reactions of the two elements. Selenium is both a good nucleophile and a good electrophile, and this property allows it to easily cycle between reduced and oxidized states without becoming permanently oxidized [2,8]. These observations lead to the hypothesis that Sec confers a redox advantage to enzymes instead of a kinetic advantage.  


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== Analogues ==
== Analogues ==


Human TrxR1 and GR share a high active site residue similarity at the cofactor and substrate binding sites as well as a similar reaction mechanism with respect to the reductive half-reaction[5]. In Sec-mTrxRs, the 8 membered selenosulfide ring acts as an internal substrate that receives electrons from the N-terminal disulfide once it is reduced by NADPH. In contrast, GR and LipDP are essentially truncated forms of mTrxR that do not contain the flexible C-terminal tail. Instead, GR and LipDH reduce external substrates such as oxidized glutathione and lipoic acid, respectively[6].
Human TrxR1 and glutathione reductase (GR) share a high active site residue similarity at the cofactor and substrate binding sites as well as a similar reaction mechanism [5]. In Sec-mTrxRs, the 8 membered selenosulfide ring acts as an internal substrate that receives electrons from the N-terminal disulfide once it is reduced by NADPH. In contrast, GR and LipDP are essentially truncated forms of mTrxR that do not contain the flexible C-terminal tail. Instead, GR and LipDH reduce external substrates such as oxidized glutathione and lipoic acid, respectively[6].  
 
 
   
   
== Mechanism ==
== Mechanism ==
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