DNA in action: Difference between revisions
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== Introduction == | == Introduction == | ||
Glutathione peroxidases are a family of enzymes that have antioxidant properties.<ref name="Koh">PMID: 17531267</ref> It fights against oxidative stress by removing reactive oxygen species (ROS) from the cell.<ref name="Koh"/> Glutathione peroxidase (GPx) can convert hydrogen peroxide to water using glutathione (2GSH + H2O2 → GS–SG + 2H2O), and can reduce peroxide radicals to their corresponding alcohol forms.<ref> Fanucchi, M.V. Chapter 11 – Development of Antioxidant and Xenobiotic Metabolizing Enzyme Systems. The Lung: Development, Aging, and the Environment, Second Edition; Harding, R., Pinkerton, K.E.; Academic Press, 2014; 223-231.</ref> To do this, GPx utilizes glutathione, glutathione reductase, and cofactors FAD and NADPH. Interestingly, GPx in plants typically rely on thioredoxin instead of glutathione as its electron donor to reduce ROS.<ref name="Koh"/> Being able to utilize both thioredoxin and glutathione as substrates is not very common, generally speaking, but it has been seen before in both thioredoxin-dependent and glutathione-dependent antioxidant systems (for example, thioredoxin reductase from Karenia brevis).<ref name="Koh"/>,<ref>Colon, R.; Wheater, M.; Joyce, E.J.; Ste.Marie, E.J.; Hondal, R.J.; Rein, K.S. The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues. J. Nat. Prod. 2021, 84 (11), 2961-2970. DOI: 10.1021/acs.jnatprod.1c00795</ref> Additionally, plants often have cysteine in their active site instead of selenocysteine found in most other GPx homologues.<ref name="Koh"/> Having cysteine in the active site typically reduces the catalytic efficiency of the enzyme in comparison with its selenocysteine-containing counterparts. Even though plant GPxs are slower, they can reduce wider variety of ROS, as they have lower substrate specificity.<ref name="Koh"/> <br /> | Glutathione peroxidases are a family of enzymes that have antioxidant properties.<ref name="Koh">PMID: 17531267</ref> It fights against oxidative stress by removing reactive oxygen species (ROS) from the cell.<ref name="Koh"/> Glutathione peroxidase (GPx) can convert hydrogen peroxide to water using glutathione (2GSH + H2O2 → GS–SG + 2H2O), and can reduce peroxide radicals to their corresponding alcohol forms.<ref> Fanucchi, M.V. Chapter 11 – Development of Antioxidant and Xenobiotic Metabolizing Enzyme Systems. The Lung: Development, Aging, and the Environment, Second Edition; Harding, R., Pinkerton, K.E.; Academic Press, 2014; 223-231.</ref> To do this, GPx utilizes glutathione, glutathione reductase, and cofactors FAD and NADPH. Interestingly, GPx in plants typically rely on thioredoxin instead of glutathione as its electron donor to reduce ROS.<ref name="Koh"/> Being able to utilize both thioredoxin and glutathione as substrates is not very common, generally speaking, but it has been seen before in both thioredoxin-dependent and glutathione-dependent antioxidant systems (for example, thioredoxin reductase from ''Karenia brevis'').<ref name="Koh"/>,<ref>Colon, R.; Wheater, M.; Joyce, E.J.; Ste.Marie, E.J.; Hondal, R.J.; Rein, K.S. The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues. J. Nat. Prod. 2021, 84 (11), 2961-2970. DOI: 10.1021/acs.jnatprod.1c00795</ref> Additionally, plants often have cysteine in their active site instead of selenocysteine found in most other GPx homologues.<ref name="Koh"/> Having cysteine in the active site typically reduces the catalytic efficiency of the enzyme in comparison with its selenocysteine-containing counterparts. Even though plant GPxs are slower, they can reduce wider variety of ROS, as they have lower substrate specificity.<ref name="Koh"/> <br /> | ||
[[Image: | [[Image:Vitamin_E_GPx_Mechanism_EJJ_5-3-2022.tif|thumb|Caption for the image]] | ||
This structure, 2P5R, is the oxidized form of glutathione peroxidase 5 from Populus trichocarpa x Populus deltoides (PtGPX5), from a paper entitled “Crystal Structures of a Poplar Thioredoxin Peroxidase that Exhibits the Structure of Glutathione Peroxidases: Insights into Redox-driven Conformational Changes”.<ref name="Koh"/> At the time of this publication, there were only six crystal structures of GPxs, all of which were mammalian.<ref name="Koh"/> This paper was ground-breaking, as it provided the first GPx structures not from mammals. Black cottonwood poplar was chosen as the model organism because at the time, its full genome had recently been released and it had six GPX genes.<ref name="Koh"/> PtGPX5 got classified as a GPx-5, the category of GPxs which are not selenoproteins.<ref name="Koh"/><br /> | This structure, 2P5R, is the oxidized form of glutathione peroxidase 5 from ''Populus trichocarpa x Populus deltoides'' (PtGPX5), from a paper entitled “Crystal Structures of a Poplar Thioredoxin Peroxidase that Exhibits the Structure of Glutathione Peroxidases: Insights into Redox-driven Conformational Changes”.<ref name="Koh"/> At the time of this publication, there were only six crystal structures of GPxs, all of which were mammalian.<ref name="Koh"/> This paper was ground-breaking, as it provided the first GPx structures not from mammals. Black cottonwood poplar was chosen as the model organism because at the time, its full genome had recently been released and it had six GPX genes.<ref name="Koh"/> PtGPX5 got classified as a GPx-5, the category of GPxs which are not selenoproteins.<ref name="Koh"/><br /> | ||
[[Image:PtGPX5 mechanism with Cys44+Cys92 disulfide EJJ 5-3-2022.tif | [[Image:PtGPX5 mechanism with Cys44+Cys92 disulfide EJJ 5-3-2022.tif]] | ||
---- | ---- | ||
< | <Structure load='2p5r' size='450' frame='true' align='left' caption='Glutathione Peroxidase 5 from poplar trees' scene='Insert optional scene name here' /> | ||
== Structural highlights == | == Structural highlights == | ||
Oxidized PtGPX5 was crystallized as a dimer in the asymmetric unit, with a resolution of 2.45Å.<ref name="Koh"/> The oxidized conformation of PtGPX5 has less defined regions than the reduced form, especially between residues 77-84, where there was barely any electron density in one of the chains.<ref name="Koh"/> As with any protein model, there is some level of interpretation. These researchers took the electron density and sequence of the flexible loop (residues 73-100) from the chain they could see and applied it to the loop containing residues 77-84 which they couldn’t see, keeping allowable Ramachandran conformations in mind.<ref name="Koh"/> This inconveniently contains Cys92, a residue involved in catalysis/disulfide bond formation.<ref name="Koh"/> They also predicted the presence of five calcium ions in the oxidized form, since it was crystallized with calcium chloride.<ref name="Koh"/> <br /> | Oxidized PtGPX5 was crystallized as a dimer in the asymmetric unit, with a resolution of 2.45Å.<ref name="Koh"/> The oxidized conformation of PtGPX5 has less defined regions than the reduced form, especially between residues 77-84, where there was barely any electron density in one of the chains.<ref name="Koh"/> As with any protein model, there is some level of interpretation. These researchers took the electron density and sequence of the flexible loop (residues 73-100) from the chain they could see and applied it to the loop containing residues 77-84 which they couldn’t see, keeping allowable Ramachandran conformations in mind.<ref name="Koh"/> This inconveniently contains Cys92, a residue involved in catalysis/disulfide bond formation.<ref name="Koh"/> They also predicted the presence of five calcium ions in the oxidized form, since it was crystallized with calcium chloride.<ref name="Koh"/> <br /> | ||
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The same dimerization pattern was observed in both the oxidized and reduced forms (which required different crystallization conditions).<ref name="Koh"/> The dimerization interfaces of both forms were about the same size and contained a 3:2 ratio of non-polar to polar atoms.<ref name="Koh"/> This dimerization interface includes the C-terminus, which is different than previously crystallized GPx structures.<ref name="Koh"/> The dimerization interface is stabilized by hydrogen bonds involving the polar side chains and van der Waals interactions between the hydrophobic and aromatic side chains.<ref name="Koh"/> <br /> | The same dimerization pattern was observed in both the oxidized and reduced forms (which required different crystallization conditions).<ref name="Koh"/> The dimerization interfaces of both forms were about the same size and contained a 3:2 ratio of non-polar to polar atoms.<ref name="Koh"/> This dimerization interface includes the C-terminus, which is different than previously crystallized GPx structures.<ref name="Koh"/> The dimerization interface is stabilized by hydrogen bonds involving the polar side chains and van der Waals interactions between the hydrophobic and aromatic side chains.<ref name="Koh"/> <br /> | ||
Mechanistically, it is proposed that Cys44 scavenges the ROS and in turn gets oxidized to sulfenic acid.<ref name="Koh"/> Then, Cys92 quickly reacts with Cys44-SOH to create a disulfide bond.<ref name="Koh"/> In the oxidized form of PtGPX5, Cys44 of the α1-helix and Cys92 of the flexible loop (α2-helix when reduced) form a disulfide bond.<ref name="Koh"/> The formation of the disulfide causes the regions the involved cysteines are in to turn towards each other, shortening the distance between the two cysteines by 12.1Å in comparison to the reduced form.<ref name="Koh"/> It is proposed that both of these cysteines can also form disulfide bonds with poplar thioredoxin’s Cys38.<ref name="Koh"/> The electron density of the active site is much better in the reduced form, but the disulfide bond is very evident in the oxidized form. Because of this, it is difficult to speculate the exact local environment around the active site, although the authors propose that nearby tryptophan residues may play a role in substrate recognition.<ref name="Koh"/> Additionally, it is known from the sequence and from the reduced form that Cys92 is in a very negatively-charged region.<ref name="Koh"/> This is interesting because this is not that case in mammalian GPxs, but further theories about its relevance cannot be deduced due to the lack of electron density in the flexible loop of the oxidized structure.<ref name="Koh"/><br /> | Mechanistically, it is proposed that Cys44 scavenges the ROS and in turn gets oxidized to sulfenic acid.<ref name="Koh"/> Then, Cys92 quickly reacts with Cys44-SOH to create a disulfide bond.<ref name="Koh"/> In the oxidized form of PtGPX5, Cys44 of the α1-helix and Cys92 of the flexible loop (α2-helix when reduced) form a disulfide bond.<ref name="Koh"/> The formation of the disulfide causes the regions the involved cysteines are in to turn towards each other, shortening the distance between the two cysteines by 12.1Å in comparison to the reduced form.<ref name="Koh"/> It is proposed that both of these cysteines can also form disulfide bonds with poplar thioredoxin’s Cys38.<ref name="Koh"/> The electron density of the active site is much better in the reduced form, but the disulfide bond is very evident in the oxidized form. Because of this, it is difficult to speculate the exact local environment around the active site, although the authors propose that nearby tryptophan residues may play a role in substrate recognition.<ref name="Koh"/> Additionally, it is known from the sequence and from the reduced form that Cys92 is in a very negatively-charged region.<ref name="Koh"/> This is interesting because this is not that case in mammalian GPxs, but further theories about its relevance cannot be deduced due to the lack of electron density in the flexible loop of the oxidized structure.<ref name="Koh"/><br /> | ||
</StructureSection> | </StructureSection> | ||
