DNA in action

== 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.<ref> Higuchi, M. Wheat and Rice in Disease Prevention and Health-Benefits, risks and mechanisms of whole grains in health promotion. Elsevier; Watson, R., Preedy, V.R., Zibadi, S.; Academic Press, 2014; 547-557.</ref> Interestingly, GPx in plants typically rely on thioredoxin instead of glutathione as its electron donor to reduce ROS.<ref name="Koh"/> <ref> Burk, R.F.; Hill, K.E. Biotransformation. ''Comprehensive Toxicology'', 2010.</ref> 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"/> <ref> Ursini, F.; Maiorino, M. Glutathione Peroxidases. ''Encyclopedia of Biological Chemistry'', Second Edition; 2013.</ref> 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: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"/><ref>PMID: PMID: 6852035</ref><ref>PMID: 9180378</ref><ref>PMID: 16054503</ref>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]]

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<Structure load='2p5r' size='750' frame='true' align='left' caption='Glutathione Peroxidase 5 from poplar trees' scene='Insert optional scene name here' />

== 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 />

PtGPX5 is structurally similar to animal GPxs, as it is a dimer and has a β-sheet core.<ref name="Koh"/> Each chain has 170 amino acids and 19.36kDa.<ref name="Koh"/> Structurally, PtGPX5 is quite similar to other GPxs, varying by about 1Å.<ref name="Koh"/> PtGPX5 is missing an N-terminal oligomerization region and a region that corresponds to the mammalian GPxs that are tetramers.<ref name="Koh"/> One notable difference between PtGPX5 and mammalian GPxs is that PtGPX5 has an overall negative charge on the protein’s surface, while mammalian GPxs are more neutral.<ref name="Koh"/><br />

Each subunit has the classic thioredoxin fold, which is a twisted β-sheet in the middle surrounded by a few α-helices.<ref name="Koh"/> Interestingly, this is not a true ten-stranded β-sheet as seen in mammalian GPxs, as there is a 6Å distance between two of the strands, preventing hydrogen bonding.<ref name="Koh"/> When the oxidized and reduced conformations are superimposed, they deviate by 4.02Å, primarily in the α1-and α2-helices.<ref name="Koh"/> In the α1-helix of the oxidized form, there is a little unwinding around catalytic Cys44, which results in a small local arrangement.<ref name="Koh"/> Then, α2-helix completely unwinds to form a long flexible loop containing another catalytic cysteine (Cys92).<ref name="Koh"/> If α1-and α2-helices are excluded, then the oxidized and reduced conformations line up very well, varying only by 0.525Å.<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 />

== Crystallization Methods ==

PtGPX5 was crystallized using selenomethionine methods to help with phasing issues.<ref name="Koh"/> Crystals of oxidized PtGPX5 were grown over two days in 0.1M Tris-HCl (pH 8.5), 25% (w/v) PEG 4000, and 0.2M calcium chloride, whereas crystals of the reduced form took a week to grow in 0.1M HEPES (pH 7.5), 0.05M cadmium sulphate hydrate, and 1.0M sodium acetate.<ref name="Koh"/> Oxidized PtGPX5 was trigonal and designated form II, meaning that it is part of the ''P''3121 space group and has cell dimensions of ''a''=71.6Å and ''c''=48.1Å.<ref name="Koh"/> There are two subunits per asymmetric unit.<ref name="Koh"/>

== Summary ==

Glutathione peroxidases are part of the antioxidant network that minimizes the buildup of ROS in a cell. PtGPX5 was the seventh GPx to be crystallized and to have the structure determined.<ref name="Koh"/> Although GPxs don’t typically use thioredoxin as a substrate, this one does.<ref name="Koh"/> It is structurally similar to animal GPxs and has a thioredoxin fold. Its dimerization interface remains conserved regardless of oxidation state and of crystallization methods.<ref name="Koh"/> It was easier to crystallize PtGPX5 in the oxidized state compared to the reduced state.<ref name="Koh"/> The greatest conformational changes occur when it is switching redox states, as the α2-helix completely unfolds.<ref name="Koh"/> It becomes a flexible loop when Cys44 gets oxidized and Cy92 subsequently forms a disulfide with it.<ref name="Koh"/>

[[Image:PtGPX5 and human GPx alignment EJJ 5-3-2022.tif]]

== References ==

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