Msrs (methionine sulfoxide reductases), MsrA and MsrB, are repair enzymes that reduce methionine sulfoxide residues in oxidatively damaged proteins to methionine residues in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. In recent years, significant efforts have been made to explore the catalytic properties and physiological functions of these enzymes. In the current review, we present recent progress in this area, with the focus on mammalian MsrA and MsrBs including their roles in disease, evolution and function of selenoprotein forms of MsrA and MsrB, and the biochemistry of these enzymes.

INTRODUCTION

ROS (reactive oxygen species), such as superoxide anion, hydroxyl radical and hydrogen peroxide, may be generated at various sites in cells during aerobic metabolism. Oxidative damage to proteins and other biomolecules by ROS has been implicated in a variety of diseases and the aging process. Both side chains of amino acid residues and the peptidic backbone of proteins can be targeted and oxidatively modified [1,2]. These modifications may be reversible or irreversible. Sulfur-containing amino acids, cysteine and methionine, are the most susceptible to oxidation, but at least some oxidized forms of these residues can be repaired in cells by specific reductase systems.

The two-electron oxidation product of cysteine, sulfenic acid, may further react with another cysteine residue to form intra- or inter-molecular disulfide bonds or be further oxidized to sulfinic acid. The disulfide can then be reduced by glutaredoxin or thioredoxin systems [3,4]. Cysteine sulfinic acids had initially been thought to be irreversible, but, at least in peroxiredoxins, they may be reduced by a recently identified protein, sulfiredoxin [5,6].

ROS can also oxidize free and protein-bound methionines, generating a diastereomeric mixture of S- and R-forms of methionine sulfoxide because of the chiral nature of sulfur in methionine sulfoxide [7]. Formation of methionine sulfoxide may lead to a significant change in 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 [8]. However, cells have evolved a mechanism to reverse methionine oxidation with a repair system that supports methionine sulfoxide reduction [912]. Msrs (methionine sulfoxide reductases) are the enzymes responsible for this function and are thus viewed as antioxidant and protein repair enzymes.

METHIONINE SULFOXIDE REDUCTASES

Msrs catalyse the reduction of free and protein-bound methionine sulfoxides to methionine. Two distinct enzyme families have evolved for the reduction of methionine sulfoxide residues in proteins (Figure 1). MsrA is specific for the S-form of methionine sulfoxide, whereas 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 absent in many hyperthermophiles and intracellular parasites [13,14]. The presence of Msr genes in anaerobic organisms (e.g. Clostridium acetobutylicum and Chlorobium tepidum) may be explained by their role in protein repair following transient exposure to oxygen. Perhaps parasites lacking Msr genes may use metabolic pathways of the host to overcome their deficiency in protein repair. Why certain other micro-organisms (e.g. Thermotoga maritima), that live at high temperatures and in the presence of oxygen do not possess Msr genes is not understood. The likely possibility is that, at high temperatures, reduction of methionine sulfoxide does not require a catalyst.

A pathway of methionine sulfoxide reduction

Figure 1
A pathway of methionine sulfoxide reduction

Free and protein-bound methionine residues can be readily oxidized by ROS to a mixture of diastereomers of methionine sulfoxide. MsrA stereospecifically reduces methionine-S-sulfoxides, whereas MsrB is specific for the R-form of methionine sulfoxide, in the presence of reducing agents, such as thioredoxin or DTT.

Figure 1
A pathway of methionine sulfoxide reduction

Free and protein-bound methionine residues can be readily oxidized by ROS to a mixture of diastereomers of methionine sulfoxide. MsrA stereospecifically reduces methionine-S-sulfoxides, whereas MsrB is specific for the R-form of methionine sulfoxide, in the presence of reducing agents, such as thioredoxin or DTT.

The first Msr, now known as MsrA, was discovered 26 years ago by Brot et al. [15] as an enzyme that could restore the biological activity of a ribosomal protein L12. This enzyme was also found to restore the activity of oxidized α-1-proteinase inhibitor by reducing methionine sulfoxides in this protein [16]. In the early 1990s, the MsrA gene was cloned from Escherichia coli [17] and later the bovine MsrA gene was also cloned [18]; the corresponding protein was found to stereoselectively reduce the S-form of methionine sulfoxide [19].

Interestingly, the other stereospecific Msr enzyme (MsrB) has only recently been identified. It has been reported that a gene coding for the PilB homologue protein from Staphylococcus aureus was located downstream of the MsrA gene, and that this protein has an Msr activity for a mixture of the R- and S-forms of methionine sulfoxide [20]. Grimaud et al. [21] found that an E. coli YeaA homologous with PilB has an Msr activity and, together with MsrA, can more fully repair oxidized methionine residues than MsrA alone. This protein was designated as MsrB and was proposed to either repair buried methionine sulfoxides or be specific for the R-form of methionine sulfoxide. Comparative genomic analyses revealed that the pattern of MsrB occurrence closely matched that of only one protein, MsrA, and this correlated evolution suggested that these two proteins are functionally linked [14]. Further biochemical studies found that mammalian and Drosophila MsrBs are zinc-containing Msrs specific for the R-form of methionine sulfoxide [14,22]. The mammalian MsrB was initially described as selenoprotein R (or selenoprotein X) and was identified computationally by searching for RNA structures required for selenocysteine insertion [23,24]. Independently other laboratories have also reported on the discovery of stereospecific activity of MsrB [25,26].

MsrA and MsrB genes cluster in many bacterial genomes and are often expressed as polycistronic mRNA [14]. Moreover, MsrA and MsrB may be directly fused to form a single polypeptide, e.g. Neisseria MsrA/MsrB fusion protein [27]. The number of MsrA and MsrB genes in different organisms is variable. Single MsrA and MsrB genes were found in organisms such as E. coli, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster [13,14]. However, multiple MsrA and/or MsrB genes may be present in some bacteria. Members of the plant kingdom, such as Arabidopsis thaliana and Chlamydomonas reinhardtii, also contain multiple MsrA and MsrB genes [13,28,29].

A recent paper [30] reported a new type of Msr from E. coli that is specific for the free form of methionine-R-sulfoxide, but is not active with the protein-bound form. Homologues of this enzyme are also present in some eukaryotes, such as S. cerevisiae, but are absent from mammals. This protein belongs to a family of proteins containing a GAF domain. It would be interesting to determine the contribution of this enzyme to the overall pathway of methionine sulfoxide reduction.

A SINGLE MsrA AND THREE MsrB GENES GENERATE ENZYMES AT MULTIPLE LOCATIONS IN MAMMALIAN CELLS

A single MsrA gene exists in human and mouse genomes (Figure 2). The most abundant MsrA form generated from this gene contains a typical N-terminal mitochondrial signal peptide, but interestingly the rat protein was found in both cytosol and mitochondria [31]. Similarly, we found that mouse MsrA containing a mitochondrial signal was targeted to the cytosol and nucleus as well as to mitochondria [32]. Structural and functional elements in this mitochondrial form appear to play a role in cellular distribution of the protein. More slowly folding and misfolded MsrA forms are targeted to mitochondria, whereas robust MsrA folding retains a significant portion of MsrA in the cytosol. However, the detailed molecular mechanism and regulation of this unusual enzyme partitioning between cellular compartments are not known. Recently, an additional MsrA form was identified, which is generated by alternative first exon splicing and resides in the cytosol and nucleus [33,34]. This MsrA isoform is enzymatically active and the corresponding mRNA was detected in monkey retina [33].

Mammalian Msrs

Figure 2
Mammalian Msrs

Mammals have one MsrA and three MsrB genes. MsrA that contains the N-terminal MTS (mitochondrial targeting signal) is targeted to the cytosol, nucleus and mitochondria. An additional variant, generated by alternative first exon splicing, was also identified. 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. The catalytic cysteine or selenocysteine residue and the recycling cysteine residues involved in the catalytic mechanisms of MsrA and MsrB are indicated. The locations of zinc-binding cysteine residues that occur in two CxxC motifs in MsrBs are also shown.

Figure 2
Mammalian Msrs

Mammals have one MsrA and three MsrB genes. MsrA that contains the N-terminal MTS (mitochondrial targeting signal) is targeted to the cytosol, nucleus and mitochondria. An additional variant, generated by alternative first exon splicing, was also identified. 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. The catalytic cysteine or selenocysteine residue and the recycling cysteine residues involved in the catalytic mechanisms of MsrA and MsrB are indicated. The locations of zinc-binding cysteine residues that occur in two CxxC motifs in MsrBs are also shown.

In contrast with a single MsrA gene, three MsrB genes have been identified in mammals [35,36]. The first known mammalian MsrB was also the first selenoprotein identified computationally [23,24]. This protein is currently known as MsrB1 [35]. The second mammalian MsrB was first reported as CBS-1, a human protein with high similarity to bacterial PilB [37]. CBS-1 was also found to have MsrB activity [38] and was designated as MsrB2. This enzyme contains a cysteine residue in place of the selenocysteine residue found in MsrB1 and an N-terminal signal peptide that targets the protein to mitochondria [35]. The third mammalian MsrB, MsrB3, also contains a cysteine residue in the active site [35]. Interestingly, human MsrB3 gives rise to two forms, MsrB3A and MsrB3B, 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, whereas MsrB3B contains a different signal peptide at the N-terminus and is targeted to mitochondria. However, studies on mouse MsrB3 found no evidence for alternative splicing [39]. Instead, mouse MsrB3 has consecutive ER and mitochondrial targeting signals at the N-terminus. This protein is targeted to the ER, and the function of the mitochondrial signal appears to be masked by the ER signal peptide. It cannot be excluded, however, that the mitochondrial signal may be activated in this protein through an unknown mechanism, at least in some cell types.

The findings that MsrA and MsrB are present in multiple cellular locations indicate that the methionine sulfoxide reduction system is maintained in different compartments in mammalian cells for repair of oxidized methionine residues (Figure 3).

Methionine sulfoxide reduction system in mammals

Figure 3
Methionine sulfoxide reduction system in mammals

MsrA and MsrBs occur in different cellular compartments and support the methionine sulfoxide reduction system in mammals by repairing oxidatively damaged proteins, protecting cells from oxidative stress and regulating protein function.

Figure 3
Methionine sulfoxide reduction system in mammals

MsrA and MsrBs occur in different cellular compartments and support the methionine sulfoxide reduction system in mammals by repairing oxidatively damaged proteins, protecting cells from oxidative stress and regulating protein function.

The fact that the three MsrB gene products are distributed in different cellular compartments implies that each protein has a role in antioxidant defence. It remains to be elucidated whether MsrBs have clear substrate specificity. Further studies are also needed to address the physiological roles of each protein with regard to their locations and to identify their targets in cells. The presence of both MsrA and MsrBs in multiple compartments is consistent with the idea that these enzymes complement each other with the exception of the ER, where only MsrB has been detected. Immunofluorescence experiments to localize either exogenous or endogenous MsrA revealed no evidence for the presence of this enzyme in the ER [32,40,41]. However, although MsrA may be missing in the ER, it is possible that the ability to reduce all methionine sulfoxides would be preserved, if this compartment has an enzyme with epimerase activity, which converts the S-form of methionine sulfoxide into the R-form, and which is then accessible to MsrB3.

PHYSIOLOGICAL ROLES OF Msrs AS ANTIOXIDANTS AND REPAIR PROTEINS

Reversible oxidation of methionine residues has been implicated in various biological and pathological processes, including oxidative stress, aging and neurodegenerative diseases [12,4246]. The primary role of MsrA and MsrB is to repair proteins damaged by oxidation. In addition, these enzymes can regulate protein function by targeting specific methionine sulfoxide residues for reduction. Finally, some surface-exposed methionine residues can be oxidized without any impact on protein function. It was proposed that such methionine residues, in combination with Msrs, function as antioxidants by scavenging ROS and therefore protecting cells from oxidative stress [47].

There are numerous reports describing the roles of Msrs in antioxidant defence. MsrA was found to protect cells against oxidative stress in several micro-organisms, including S. cerevisiae, S. aureus, Neisseria gonorrhoeae and Helicobacter pylori [4851]. This protein was also found to play an important role in the viability of lens cells [52,53], retinal pigmented cells [54] and human WI-38 fibroblasts [41] by providing resistance to oxidative stress. In addition, MsrA was found in human skin and up-regulation of the protein was observed in response to UV irradiation and hydrogen peroxide, suggesting a role of MsrA in photoprotection in epidermis [55,56]. There is also direct evidence that MsrA can decrease the levels of carbonylation in proteins, which is one of the markers of oxidative stress in cells [57], by alleviating cellular oxidative stress. The antioxidant role of MsrBs has been previously studied in lens cells [58], showing that all three MsrBs (MsrB1, MsrB2 and MsrB3) could protect against oxidative stress in the lens cells.

Msrs IN AGING

Accumulation of oxidatively damaged proteins produced by the action of ROS is thought to be one of the major causes of aging. It is easily predicted that Msr enzymes might be directly involved in the aging process; hence, their roles in aging and regulation of lifespan have been investigated. Indeed, knockout of the MsrA gene in mice reduced lifespan by approx. 40% [59]. Moreover, overexpression of bovine MsrA in Drosophila extended lifespan by 70%, as well as increased resistance to paraquat-induced oxidative stress [60]. Total Msr activity was found to be reduced during aging in rat kidney and liver [61] and the expression levels of MsrA and MsrB2 were decreased in senescent WI-38 fibroblasts compared with control cells [40].

Since MsrBs probably account for half of the total methionine sulfoxide reduction, they may play a role similar to MsrAs in delaying the aging process. However, thus far this role has only been studied in a lower eukaryote, S. cerevisiae [62]. Overexpression of MsrB extended yeast lifespan under caloric restriction conditions, but not in cells grown in regular medium where MsrA instead could regulate lifespan. It was also found that under ROS-deficient, anaerobic growth conditions, neither MsrA nor MsrB affected the lifespan of yeast cells. To understand the precise role of MsrB in the aging process in higher organisms, studies are needed using knockout and transgenic animal models. In particular, since mammals have three MsrBs, targeting multiple MsrB genes may be required to determine the contribution of individual MsrB genes and their possible synergic effects on the regulation of the aging process. In addition, the studies with combined MsrA and MsrB deficiency and overexpression in animals may provide insights into the role of methionine sulfoxide reduction in aging. Finally, it would be interesting to determine which cellular compartments and tissues are responsible for the lifespan extension provided by Msrs, and which proteins are targeted by these enzymes in young and aging cells.

Msrs AND NEURODEGENERATIVE DISORDERS

There is growing evidence that the Msrs have a role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. MsrA activity is decreased in the brains of Alzheimer's disease patients [63]. β-Amyloid deposition is thought to cause the neurotoxicity of Alzheimer's disease. It appears that oxidation of Met35 in β-amyloid is critical for aggregation and neurotoxicity of the protein [44,64]. A recent study reported that MsrA-knockout mice have an enhanced neurodegeneration in the hippocampus and that this region of the brain is characterized by elevated levels of β-amyloid deposition and damaged astrocytes [65]. Aggregation of α-synuclein is critical for the pathogenesis of Parkinson's disease, and oxidation of this protein at methionine residues has been suggested to be involved in its fibrillation [66]. Mutations in another protein, DJ-1, is associated with an early-onset form of Parkinson's disease, which is an autosomal recessive defect. In addition to genetic alterations in DJ-1, oxidative stress can directly affect this protein. A recent study revealed that oxidative modifications of DJ-1 are associated with sporadic Parkinson's and Alzheimer's diseases [67]. It was found that DJ-1 was oxidatively modified in the brains of patients. In particular, oxidation of Met133 and Met134 to methionine sulfoxide or sulfone may be a modification unique to Parkinson's disease.

TARGET PROTEINS FOR Msrs

As discussed above, oxidation of methionine residues can affect biological activities of proteins and reduction of methionine sulfoxides back to methionine residues can restore these activities. Thus reversible methionine oxidation may regulate protein function. Ribosomal protein L12 [15], α-1-proteinase inhibitor [16], calmodulin [68], Fft (a prokaryotic signal recognition particle component) [69], HIV-2 protease [70] and shaker potassium channel [71] have previously been described as substrates for Msr proteins, whose functions are impaired by oxidation of methionine residues and restored by the activity of Msr enzymes. It is expected that there are many additional Msr targets in cells; however, methodological difficulties precluded identification of such proteins on a large scale.

It has been reported that both MsrA and MsrB are able to reduce methionine sulfoxide residues in calmodulin [21,72], and that each enzyme can repair four to six out of eight methionine sulfoxide residues initially present, whereas a combination of MsrA and MsrB can fully repair the oxidized methionine residues [21]. However, a recent study reported that methionine sulfoxide at position 124 is differentially repaired by MsrA and MsrB [73]. MsrB reduced on average 25% of this methionine sulfoxide (corresponding to half the amount of the R-form), whereas MsrA repaired 50% of methionine sulfoxide (the entire amount of the S-form). Other methionine sulfoxide residues were found to be fully reduced by each enzyme.

Msrs AS SELENOPROTEINS

Selenium, an essential trace element in mammals, is inserted co-translationally into proteins in the form of selenocysteine [74,75]. The selenocysteine-containing proteins, selenoproteins, occur in organisms in all three domains of life. Of the selenoproteins characterized thus far, most are oxidoreductases, such as formate dehydrogenase [76], glutathione peroxidase [77] and thioredoxin reductase [78], in which selenocysteine occupies the active sites. MsrB1, a recent addition to the group of selenium-containing oxidoreductases, has selenocysteine in the position that corresponds to catalytic cysteine in other MsrBs.

The selenoprotein form of MsrB has only been described in vertebrate animals. However, we recently found that this MsrB form also occurs in some invertebrates [79]. In the case of MsrA, selenoprotein forms have been identified in bacteria, algae and invertebrate animals [29,80]. Interestingly, the distribution of selenoprotein forms of MsrA and MsrB is different, suggesting an independent origin of these proteins.

To examine the role of selenocysteine in catalysis by MsrA and MsrB, mammalian selenoprotein MsrB1 was expressed in E. coli with the help of a bacterial SECIS (selenocysteine insertion sequence) element that was designed immediately downstream of the UGA selenocysteine codon [35]. This selenoprotein had an 800-fold higher activity than the corresponding cysteine form, indicating a critical role for selenocysteine in the catalytic function of this enzyme. Selenoprotein MsrB1 was also examined in mammalian cells [81] and found to have a 100-fold higher activity than the cysteine mutant form. A high catalytic activity was also observed in selenocysteine-containing MsrA from Chlamydomonas [80]. This enzyme exhibited 10-fold higher activity than its cysteine mutant and, in fact, was the most efficient MsrA catalyst known. Taken together, these studies provided evidence for catalytic advantages of selenocysteine in Msrs and probably other thiol-dependent oxidoreductases.

Multiple sequence alignments reveal that different sets of active-site features have evolved in selenoprotein and non-selenoprotein MsrBs. Three conserved residues (His77, Val/Ile81 and Asn97; numbering is based on the mouse MsrB1 sequence) are uniquely conserved in cysteine-containing proteins, but are absent from selenoprotein forms. The corresponding residues in MsrB1 are Gly77, Glu81 and Phe97 respectively. It has been found that the three conserved residues in cysteine-containing MsrB2 and MsrB3 are critical for enzyme function, but introducing these residues into selenoprotein MsrB1 was detrimental to the activity of this enzyme [81]. These data implied that distinct sets of active-site features maximize catalytic efficiencies in selenocysteine- and cysteine-containing MsrBs.

STRUCTURES AND CATALYTIC MECHANISMS OF Msrs

MsrA

Crystal structures for MsrAs from E. coli [82], Bos taurus [83], Mycobacterium tuberculosis [84] and Populus trichocarpa [85] have been reported. The MsrA fold belongs to an α/β class of proteins. The central feature of the MsrA structure is an α/β plaits motif, which includes a conserved active-site GCFWG sequence. The catalytic mechanism of MsrA is characterized by sulfenic acid chemistry and consists of three steps (Figure 4) [8689]: (i) nucleophilic attack by the catalytic cysteine residue (CysA) on the sulfoxide moiety of the substrate leading to formation of a sulfenic acid intermediate and a concomitant release of methionine; (ii) attack by the recycling cysteine residue (CysB) on the sulfenic acid intermediate to form an intramolecular disulfide bond; and (iii) reduction of the CysA–CysB disulfide bond in the enzyme by thioredoxin in vivo or DTT (dithiothreitol) in vitro leading to the fully reduced active site. The recycling CysB is located at the C-terminal region, and the catalytic CysA at the N-terminal.

General catalytic mechanisms of MsrA and MsrB

Figure 4
General catalytic mechanisms of MsrA and MsrB

Although different in sequence and structure, MsrA and MsrB have a very similar reaction mechanism. In the first step (I), the substrate, methionine sulfoxide (i.e. Met-S-SO or Met-R-SO), is reduced stereospecifically by MsrA or MsrB. The catalytic cysteine, CysA (shown in red), of MsrA or MsrB attacks the substrate to form methionine and itself is converted into a sulfenic acid intermediate. The following steps (II and III) are to regenerate the enzymes. The recycling CysB (shown in blue) attacks the sulfenic acid intermediate on CysA to form a disulfide bond, and the resulting CysA–CysB disulfide bond is further reduced by thioredoxin (Trx). DTT can be used in vitro as a reductant in place of Trx.

Figure 4
General catalytic mechanisms of MsrA and MsrB

Although different in sequence and structure, MsrA and MsrB have a very similar reaction mechanism. In the first step (I), the substrate, methionine sulfoxide (i.e. Met-S-SO or Met-R-SO), is reduced stereospecifically by MsrA or MsrB. The catalytic cysteine, CysA (shown in red), of MsrA or MsrB attacks the substrate to form methionine and itself is converted into a sulfenic acid intermediate. The following steps (II and III) are to regenerate the enzymes. The recycling CysB (shown in blue) attacks the sulfenic acid intermediate on CysA to form a disulfide bond, and the resulting CysA–CysB disulfide bond is further reduced by thioredoxin (Trx). DTT can be used in vitro as a reductant in place of Trx.

MsrAs can be divided into two groups with regard to the involvement of the recycling cysteine residue in the catalytic reaction. Group I MsrA proteins, such as the enzymes from E. coli and B. taurus, have an additional recycling cysteine residue (CysC) at the C-terminal region. Thus two recycling cysteine residues are involved in the catalytic mechanism. CysC attacks CysB of the CysA–CysB disulfide bond to form a new CysB–CysC disulfide bond, which is then reduced by thioredoxin. A recent NMR structure of oxidized E. coli MsrA (CysA–CysB disulfide form) revealed that high flexibility of the C-terminal part of the oxidized form favours the CysB–CysC disulfide bond [90]. On the other hand, a crystal structure of plastid MsrA from P. trichocarpa suggested that CysC first forms a disulfide bond with the catalytic CysA and then the disulfide bond formation between CysC and CysB occurs via thiol/disulfide exchange [85]. Group II MsrA proteins, such as the enzyme from M. tuberculosis, include proteins that do not have CysC. Only CysB is involved in the recycling of the sulfenic acid intermediate.

It is noteworthy that some MsrAs lack any recycling cysteine residues. For example, all selenoprotein MsrAs identified thus far do not have a candidate recycling cysteine residue [80]. It has also been found that the recycling cysteine residue is absent from Chlamydomonas selenoprotein MsrA and that this residue is not required for the catalytic function of the cysteine mutant (U20C) of this enzyme [80]. In the case of a protein without the recycling cysteine residue, a sulfenic acid (or selenenic acid) intermediate may be directly reduced by thioredoxin or by an as yet uncharacterized reducing agent.

MsrB

To date, two MsrB structures, for enzymes from N. gonorrhoeae [26] and Bacillus subtilis (PDB code 1XM0), are available. Interestingly, the MsrB fold is unrelated to that of MsrA, but the comparison of MsrB and MsrA structures reveals a mirror-like relationship of their active sites [26]. Although MsrB and MsrA structures are different, their catalytic mechanisms are very similar (Figure 4) [22,26,91]. Approx. 60% of MsrBs contain a recycling cysteine residue in the middle of their sequences, whereas the remaining 40%, including all three mammalian MsrBs, do not have this cysteine residue. It has been found that mammalian MsrB2 and MsrB3 do not require a recycling cysteine residue (i.e. the sulfenic acid intermediate could be directly reduced by thioredoxin), whereas selenoprotein MsrB1 uses an alternative cysteine residue in the N-terminal region as the recycling residue [81] (see Figure 2). It has been reported that Xanthomonas campestris MsrB reduces the sulfenic acid intermediate with yet another recycling cysteine residue [92]. Thus both MsrA and MsrB can use alternative mechanisms to generate the reduced form of their catalytic cysteine or selenocysteine.

Many MsrBs, including all three mammalian enzymes, contain a zinc atom. Two CxxC (x indicates any amino acid) motifs occur in these proteins to provide thiolate ligands for the metal (see Figure 2). Zinc is suggested to play a structural function in MsrB [22,93], but the precise role of this metal remains to be elucidated. Structures of zinc-containing MsrBs may help us to better understand the role of zinc in MsrB function.

Thioredoxin is generally considered as an in vivo reducing agent for MsrA and MsrB. However, a recent study revealed that thionein, a low-molecular-mass cysteine-rich protein, can serve as a reductant for both MsrA and MsrB [94]. Furthermore, selenium compounds such as selenocystamine were found to also act as reducing agents for MsrB2 and MsrB3 [95]. Further studies are needed to determine whether thionein and selenium compounds function as physiological reductants in vivo.

KINETIC CHARACTERIZATION OF Msrs

The kinetic mechanisms for both classes of Msrs are of the Ping Pong type [86,96]. For both MsrA and MsrB from Neisseria meningitidis, the thioredoxin-recycling process was found to be the rate-limiting step, whereas the formation of the sulfenic acid intermediate is very rapid and the rate of this step governs that of intradisulfide bond formation, whose rate is also high [86,88,91]. The rates of methionine formation, equivalent to the rate of the formation of the sulfenic acid intermediate, under single turnover conditions were determined to be 790 s−1 and 85 s−1 for N. meningitides MsrA and MsrB respectively [86]. These values are 230- and 80-fold higher than kcat values determined under steady-state conditions for MsrA and MsrB (3.4 s−1 and 1.1 s−1 respectively). The rates of disulfide bond formation were at least 500 s−1 and 80 s−1 for MsrA and MsrB respectively [86]. Thus the rate of formation of the disulfide bond is limited by that of sulfenic acid intermediate formation. The rate of the reduction of the disulfide bond in the thioredoxin-recycling process can be determined by measuring the rate of formation of oxidized thioredoxin. The determined rates of the formation of oxidized thioredoxin were 50 s−1 and 5 s−1 for MsrA and MsrB respectively, which are 15- and 5-fold higher respectively than kcat values under steady-state conditions [86].

CATALYTIC EFFICIENCIES OF SELENOCYSTEINE- AND CYSTEINE-CONTAINING ENZYMES

The catalytic efficiency, kcat/Km, of selenocysteine-containing MsrB1 is at least 100-fold higher than that of its cysteine mutant [35,81]. However, the cysteine-containing homologues MsrB2 and MsrB3 have catalytic efficiencies only slightly lower than that of selenoprotein MsrB1 [35,81]. In the case of selenocysteine-containing MsrA from Chlamydomonas, it has a 40-fold increased catalytic efficiency compared with its cysteine mutant [80]. Moreover, this catalytic efficiency is 40-fold higher than that of the cysteine-containing mouse MsrA [32].

REGULATION OF Msrs

Mammalian MsrA is a ubiquitous enzyme, but it occurs in tissues at different expression levels. The highest MsrA expression has been reported in kidney, liver and cerebellum [18,97]. Mammalian MsrB1, MsrB2 and MsrB3 are also expressed ubiquitously. MsrB1, like MsrA, is highly expressed in detoxification organs, such as liver and kidney (H. Y. Kim and V. N. Gladyshev, unpublished work). MsrB2 and MsrB3 are expressed at elevated levels in heart and skeletal muscle [38,98].

It has been reported that a calcium phospholipid-binding protein binds to the yeast MsrA promoter and enhances protein expression [99]. Interestingly, yeast cytosolic thioredoxins were found to be involved in transcriptional regulation of the MsrA gene [100]. Recently, promoter analysis has shown that the human MsrA gene has a TATA-less promoter and a negative regulatory cis-element recognized by a putative transcriptional factor(s) [101]. In addition, yeast glutathione peroxidase 3 was found to directly interact with MsrA by forming a disulfide bond and this interaction may regulate MsrA activity in a redox manner [102]. Transcriptional regulation of human MsrB1 has also been examined [103]. This study revealed that an Sp1 transcriptional factor may play an important role in MsrB1 expression, and that epigenetic modifications (e.g. methylation) can control the promoter activity. In MsrA-knockout mice, MsrB1 expression declined compared with that in wild-type, suggesting that MsrA may have a role in MsrB expression [104]. This study also revealed that a selenium-deficient diet enhances the level of protein oxidation and decreases MsrB1 expression. It was also reported that the expression level of MsrB1 is highly regulated by dietary selenium in transgenic TGFα (transforming growth factor)/c-myc mice [105]. A selenium-deficient diet reduced MsrB1 protein levels and activity.

Overall, some progress has recently been made with regard to regulation of Msrs; however, the details of regulation of this important class of proteins are not known. In particular, the fact that MsrB1 is a selenoprotein provides an opportunity to regulate the overall capacity for methionine sulfoxide reduction by dietary selenium.

CONCLUDING REMARKS

Msrs play a pivotal role in defending cells from oxidative stress and have been implicated in regulation of the aging process and progression of neurodegenerative diseases. Recent progress in identifying new classes of these enzymes and exploring their catalytic functions and phenotypes associated with their deficiency has provided important insights into the functions of Msrs. Selenocysteine-containing forms of both MsrA and MsrB have been identified, and these are superior catalysts compared with cysteine-containing forms. In particular, the major mammalian MsrB, MsrB1, is a selenoprotein, suggesting a possibility of regulation of the overall Msr function by dietary selenium. Many important questions regarding Msr functions remain such as: (i) the role of MsrB in the aging process; (ii) the precise roles of Msrs in neurodegenerative disorders; (iii) regulation of the methionine sulfoxide reduction pathway; and (iv) the identity of cellular targets for Msrs. Development of appropriate animal models and methodology should contribute to addressing these and other questions.

This work was supported by KOSEF (Korea Science and Engineering Foundation) grants (R13-2005-005-01004-0 and M10642040001-06N4204-00111; to H.-Y. K.), by the Yeungnam University research grants in 2006 (to H.-Y. K.) and by the NIH (National Institutes of Health) grant AG021518 (to V. N. G.).

Abbreviations

     
  • DTT

    dithiothreitol

  •  
  • ER

    endoplasmic reticulum

  •  
  • Msr

    methionine sulfoxide reductase

  •  
  • ROS

    reactive oxygen species

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