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Methionine Sulfoxide Reductase

Methionine (Met) and cysteine are sulfur-containing amino acids which are present in proteins and are essential for life. Methionine exists in form of two isomers, L-and D-methionine and L-form predominates in nature. Most plants, fungi, and bacteria can synthesize methionine from carbohydrates, organic or inorganic nitrogen, and sulfur sources. However, methionine is essential for animals, including humans and animals depends on dietary provided methionine sources [ 1, 2 ].

Both free Met and protein Met residues can be oxidized by reactive oxygen (such as superoxide anion, hydroxyl radical, hydrogen peroxide etc.) species to methionine sulfoxide (MetO). Nonenzymatic oxidation leads to formation of two diastereomers, methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO), as result of the asymmetric nature of the sulfur atom in the methionine [ 3 ]. Formation of methionine sulfoxide may significantly affect protein structure and function. In addition, methionine sulfoxide residues may be further targeted by ROS to produce methionine sulfone or radicals and to propagate oxidative damage [ 4 ]. That is why organisms have evolved a mechanism to reduce oxidised methionines. Methionine sulfoxide reductases (Msrs) are the enzymes antioxidant and protein repair enzymes that are responsable for reduction of methionine sulfoxide. Two distinct enzyme families are involved in reduction of methionine sulfoxide residues in proteins. MsrA is specific for the S-form of methionine sulfoxide and MsrB can only reduce the R-form. Msr genes are found in most organisms from bacteria to humans, even in the species that live under anaerobic conditions, but are frequently absent in hyperthermophiles and intracellular parasites [ 5, 6, 7, 8, 9, 10 ].

Methionine metabolism in mammals and methionine sulfoxide reductases system. Methionine residues can be oxidized to Methionine sh=ulfoxide by ROS. Msrs can reverse the methionine oxidation, leading to replenishment of the methionine pool. BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine beta-synthase; CSE, cystathionine-lyase; MAT, methionine S-adenosyltransferase; MetO, methionine sulfoxides; MS, methionine synthase; MT, methyltransferases; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocys-teine hydrolase; SAM, S-adenosylmethionine. [ 11, 12 ].

There are 80 solved methionine sulfoxide reductase structures in Protein Data Bank at this time (January 2016).

Crystal structure of Mycobacterium Tuberculosis methionine sulfoxide reductase A.

Crystal structure of Mouse methionine sulfoxide reductase B1 (SelR). Zinc atom is shown as pink sphere, selenocysteine is shown by red color.

Methionine S-sulfoxide reductase (MsrA) was discovered in 1980 as an enzyme that could restore the biological activity of a ribosomal protein L12 and alpha-1-proteinase inhibitor by reducing of oxidiszed methionine [ 13 ]. In contrast to MsrA, methionine R-sulfoxide reductase (MsrB) was discovered 15 years ago by comparative genomics aproache which shown that pattern of MsrB occurrence closely matched to occurrence of MsrA, that these proteins are frequently clustered in bacterial genomes and frequently forms domain fusions. Such observation suggested that these two proteins are functionally linked and MsrB function was experimentally confirmed [ 9 ]. Mammalian MsrB or selenoprotein R (SelR) was identified computationally by searching for selenoproteins in mammalian genomes [ 14 ].

Single MsrA and MsrB genes were found in organisms such as E. coli, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. Multiple MsrA and/or MsrB genes may be present in some bacteria and archaea. Unicellular and vascular plants such as Arabidopsis thaliana and Chlamydomonas reinhardtii, contain multiple MsrA and MsrB genes [ 7, 15, ]. MsrA gene is represented by single copy in human and mouse genomes. Mammalian MsrA contains N-terminal mitochondrial signal peptide. Additional MsrA form is generated by alternative first exon splicing and resides in the cytosol and nucleus [ 16, 17, 18 ].

Different cellular compartmentalization of Msrs in mammalian cells [ 11 ].

Domain structure and localization of mamalian Msrs. Mammals have one MsrA and three MsrB genes. MsrA contains the N-terminal MTS (mitochondrial targeting signal) is targeted to the cytosol, nucleus and mitochondria. Among three MsrBs, MsrB1 contains selenocysteine (U) in the catalytic site and is present in the cytosol and nucleus. MsrB2 has a typical MTS and localizes to the mitochondria. Human MsrB3 occurs in two forms, MsrB3A and MsrB3B, owing to alternative first exon splicing, and the two proteins have an N-terminal ERTS (ER targeting signal) and MTS respectively. Both proteins also have an ER RS (retention signal) consisting of a tetrapeptide at the C-terminus. MsrB3A resides in the ER, whereas MsrB3B is a mitochondrial form. However, only a mouse form of MsrB3 has been found that contains an ERTS followed by MTS and is targeted to the ER. [ 19 ].

There are three MsrB paralogs are present in mammal genomes. The first known mammalian MsrB (MsrB1 or SelR) was the first selenoprotein identified by computational aproach [ 9, 14, 20 ]. The second mammalian MsrB, MsrB2, contains a cysteine residue in place of the selenocysteine residue and an N-terminal signal peptide that targets the protein to mitochondria. The third MsrB, MsrB3, The third mammalian MsrB, MsrB3, also contains a cysteine residue in the active site [ 20, 21 ]. Human MsrB3 is represented by two forms, MsrB3A and MsrB3B, formed by alternative first exon splicing. MsrB3A contains an ER (endoplasmic reticulum) signal peptide at the N-terminus and an ER retention signal at the C-terminus, and is targeted to the ER. MsrB3B contains a different signal peptide at the N-terminus and is targeted to mitochondria. Mouse MsrB3 has ER and mitochondrial targeting signals at the N-terminus [ 22 ].

Recycling of Methionine Sulfoxide Reductases

Thioredoxins (Trxs) and glutaredoxins (Grxs) are structuraly similar thiol oxidoreductases that are involved in methionine sulfoxide reductases catalitic cycle. Thioredoxins are the major reductants for MsrA and MsrB proteins. Glutaredoxins can serve as a reductant for 1-Cys Clostridium MsrA (54). Moreover, Grx can reduce 3-Cys mouse MsrA containing resolving Cys with reduction ability comparable to that of Trx [ 23, 24, 25, 26 ]. Glutaredoxins can also be used for the reduction of 2-Cys form of MsrB as well as the 1-Cys form. In addition, Grx is more efficient reductant for 1-Cys MsrB than Trx and whereas Trx is more efficient for the 2-Cys form than Grx [ 26, 27, 28 ]. Zinc-containing cysteine rich metallothionein may serve a reductant for both MsrA and MsrB [ 29 ].

Reducing systems for regeneration of Msrs. In thioredoxin (Trx) system, electrons are transferred from NADPH to Trx via Trx reductase (TR) to reduce oxidized Msrs. In the glutaredoxin (Grx) system, electrons are transferred from NADPH to Grx via GSH to reduce oxidized Msrs. [ 11 ].

In 2-Cys MSRA and 1-Sec/1-Cys MsrB the reduction of methionine sulfoxide resulting in formation of sulfenic acid or selenenic acid on the catalytic residue, Cys or Sec (1) followed by the formation of disulfide (or selenenylsulfide) and the release of one molecule of water (2). The disulfide bond is then reduced by Trx through disulfide exchange, and a transient intermolecular covalent complex formation (3) and subsequent reduction, leading to the release of the reduced form of MSR and the oxidized form of Trx (4). For 3-Cys MSRA the first two steps are similar to 2-Cys MSRA and 1-Sec/1-Cys MsrB. A second resolving Cys reduces the first disulfide bond, leading to the formation of a disulfide bond between the two resolving Cys (3), which is then reduced by Trx (4) leading to the release of the reduced 3-Cys MSRA and oxidized Trx (5) [ 23, 30, 31 ].

Regeneration mechanisms of different forms of Msrs [ 30 ].

Methionine Sulfoxide Reductases and Signaling

Protein amino acid modification is a major mechanism for regulation of protein function. Oxidation of protein methionines may influence biological activities of proteins. The reduction of corresponding methionine sulfoxides by Msrs can regulate such activities. Futhermore, oxidation and reduction of methionines both are contributing in signaling activation. MsrA was first identified as an enzyme that could restore the activity of ribosomal protein L12 [ 9 ]. Other MsrA substrates include alpha-1-proteinase inhibitor, calmodulin, apolipoprotein A-I, inhibitor of Kappa B-alpha, TRPM6 magnesium channel and the shaker potassium channel, whose functions are damaged by methionine oxidation and restored by MsrA. Calcium-/calmodulin-dependent protein kinase II (CaMKII) is activated by calcium, but can also be activated in an ROS-dependent manner when oxidized at Met-281 and Met-282 in the regulatory domain of the protein. MsrA was found to reverse the activation of CaMKII caused by oxidation. Recent study show that flavin-containing monooxygenase containing mical proteins can stereospecifically oxidize two conserved Met residues in actin and regulate actin polymerization [ 32, 33, 34, 35, 36 ].

Substrates of methionine sulfoxide reductases. Functions of these proteins are regulated by reversible oxidation of methionines [ 34, ].

Physiological Roles of Methionine Sulfoxide Reductases

Methionine oxidation and reduction is associated with variety of biological and pathological processes, including cellular signaling, oxidative stress, and aging [ 10, 37 ]. The major role of Msrs is related to repairing of oxidatively damaged proteins. Oxidation of the cellular methionine poole is a mechanism of ROS scavenging, thus, cyclic methionine oxidation/reduction is important antioxidant defense mechanism [ 38 ]. In addition, Msrs are able to regulate the function of proteins by modulating specific methionine sulfoxide residues involved in activation or inactivation of proteins as described above [ 32, 33, 34, 35, 36 ].

Overexpression of MsrA in Drosophila is accosiated with increase of lifespan [ 39 ]. Knockout of MsrA in yeast and nematodes shortens the lifespan of these organisms [ 40, 41 ]. The effect of MsrA deletion in mice is not clear. One study reported that deletion of MsrA decreased lifespan in mice by 40% [ 42 ]. and another study showed that MsrA knockout in mice did not alter lifespan [ 43 ].

MsrB deletion and overexpression in yeast has no effect on the lifespan under normal conditions [ 44 ]. Overexpression of mouse cytosolic selenoprotein MsrB1, mitochondrial MsrB2, and Drosophila cytosolic MsrB had no influence on the lifespan of Drosophila [ 45, 46 ]. Hovewer, expression of human ER localized MsrB3A is significantly extend the lifespan of Drosophila [ 47 ]. In addition, several groups suggest that Msrs are involved in neuroprotection and Msrs defficiency is associated with neurodegeneration [ 48 ]. Finally, only oxidative stress defence role of methionine sulfoxide reductases is clearly confirmed to date.

Free Methionine Sulfoxide Reductases

New type of Msr, free methionine sulfoxide reductase (fRMsr), was identified in E. coli in 2007. This enzyme only reduces the free form of methionine-R-sulfoxide and has no activity toward the protein-based form of methionine sulfoxide [ 49 ]. fRMsr is a a GAF domain containing ezyme. Such domain is typically present in cyclic GMP phosphodiesterases, but does not bind cGMP. fRMsr GAF domain contain unique and fully conserved catalytic cysteines. fRMsr is the main enzyme responsible for the reduction of free Met-R-sulfoxide. At the same time, MsrA is responsible for the reduction of both free and protein-based Met-S-sulfoxide. fRMsrs are found in unicellular organisms, including yeast, but not in multicellular organisms [ 50 ].

Crystal structure of Saccharomyces cerevisiae free methionine-R-sulfoxide reductase.

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