Review Article The discovery of methionine sulfoxide reductase enzymes: An historical account and future perspectives

Cesare Achilli Annarita Ciana Giampaolo Minetti*

Laboratories of Biochemistry, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

Abstract L-methionine (L-Met) is the only sulphur-containing proteinogenic amino acid together with cysteine. Its importance is highlighted by it being the initiator amino acid for protein synthesis in all known living organisms. L-Met, free or inserted into proteins, is sensitive to oxidation of its sulfide moiety, with formation of L-Met sulfoxide. The sulfoxide could not be inserted into proteins, and the oxidation of L-Met in proteins often leads to the loss of biological activity of the affected molecule. Key discoveries revealed the existence, in rats, of a metabolic pathway for the reduction of free L-Met sulfoxide and, later, in Escherichia coli, of the enzymatic reduction of LMet sulfoxide inserted in proteins. Upon oxidation, the sulphur

Keywords: selenoproteins; methyl-sulfoxide; asymmetric center; enantiospecificity

redox

balance;

1. The Beginnings: Oxidation and Enzymatic Reduction of Methionine L-methionine (L-Met) was discovered by Mueller in 1923 [1], and it is the only standard a-amino acid with a sulfide (or sul-

Abbreviations: : L-Met, L-methionine; Cys, cysteine; Sec, selenocysteine; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; DTT, dithiothreitol; DABS, 4-(dimethylamino)azobenzene-4-sulfonate; rpHPLC, reverse-phase high performance liquid chromatography; SECIS, Sec insertion sequence; FMOC, N-(9-fluorenyl-methoxycarbonyl) chloride; TLC, thin-layer chromatography; NMR, nuclear magnetic resonance; Trx, thioredoxin; TrxR, thioredoxin reductase; DTE, dithioerythritol C 2015 International Union of Biochemistry and Molecular Biology V Volume 41, Number 3, May/June 2015, Pages 135–152 *Address for correspondence: Giampaolo Minetti, PhD, Department of Biology and Biotechnology “Lazzaro Spallanzani”, Laboratories of Biochemistry, University of Pavia, via Bassi, 21, 27100 Pavia, Italy. Tel.: 139 0382 987891; Fax: 138 0382 987240; E-mail: [email protected] Received 23 February 2015; accepted 19 April 2015 DOI 10.1002/biof.1214 Published online 12 May 2015 in Wiley Online Library (wileyonlinelibrary.com)

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atom becomes a new stereogenic center, and two stable diastereoisomers of L-Met sulfoxide exist. A fundamental discovery revealed the existence of two unrelated families of enzymes, MsrA and MsrB, whose members display opposite stereospecificity of reduction for the two sulfoxides. The importance of Msrs is additionally emphasized by the discovery that one of the only 25 selenoproteins expressed in humans is a Msr. The milestones on the road that led to the discovery and characterization of this group of antioxidant C 2015 BioFactors, enzymes are recounted in this review. V 41(3):135–152, 2015

phide) moiety. The sulfide may be easily oxidized to the sulfoxide (or sulphoxide, also named sulfinyl or sulphinyl), in which the sulphur atom is bound to an oxygen atom and its oxidation state changes from 22 to 0. Stronger pro-oxidant conditions lead to the formation of the sulfone (or sulphone, also named sulfonyl or sulphonyl), in which the sulphur is bound to two oxygens, or of the sulfoximine (sulphoximine), in which the sulphur atom is bound to one oxygen and one nitrogen [2]. In both cases the oxidation state of the sulphur atom becomes 12. In addition to the above mentioned chemical modifications, L-Met can undergo one-electron oxidation of its sulfide group, mediated by radical oxidative agents, that yields a very reactive radical derivative [3], or to cyclization by treatment with sodium hypoiodite, that yields dehydromethionine, which can be easily converted to L-Met sulfoxide [L-Met-(O)] after acidic treatments [2] (for the chemical structures of these molecules, Fig. 1). The oxidation of L-Met to its sulfoxide results in the introduction of a stereogenic centre at the level of the sulphur, in addition to that already present on the a-carbon that, like in all other naturally-occurring proteinogenic amino acids (except for glycine, which is not chiral) has the L configuration

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FIG 1

Chemical structure of L-methionine (L-Met) (1). Mild oxidation of L-Met (e.g. by hydrogen peroxide) produces the two diastereoisomers L-Met-S-(O) (2) and L-Met-R-(O) (3). Oxidation under stronger conditions (e.g. by hydrogen peroxide plus molybdate salts) generates L-Met sulfone (4), or, by means of reactive nitrogen species (e.g. hydrazoic acid), sulfoximine (5). A radical derivative of L-Met (6) is produced by action of hydroxyl radicals. The cyclic derivative dehydromethionine is generated by treatment with hypoiodite salt (7).

(Fischer and Rosanoff convention), which corresponds to the S absolute configuration in the Cahn Ingold Prelog naming system [the same as in all other amino acids except for cysteine (Cys) and selenocysteine (Sec), which have a R a-carbon]. Therefore, L-Met oxidation implies the formation, generally in equal amounts, of two stable diastereoisomers: L-Met-S-(O) and L-Met-R-(O), that are, respectively, the dextrorotatory and the levorotatory stereoisomers [4]. The optical stability of LMet-S-(O) and L-Met-R-(O), as well as that of almost all sulfoxides, is based on the fact that the pyramidal inversion of sulphur requires high activation energy, and therefore only takes place at high temperatures (generally 200 8C) [5]. The oxidation of free L-Met to L-Met-(O) by chemical agents, such as iodine, iodate and hydrogen peroxide, has been described since 1938 [6–8]. L-Met residues inserted in a polypeptide chain can also be oxidized to the sulfoxide [9] but, unlike what occurs for free L-Met, a certain level of specificity in the yield of the two stereoisomers was observed, such as during the treatment of calmodulin by certain oxidizing agents, where a significant excess of L-Met-R-(O) was detected [10]. Subsequently, oxidized L-Met residues in proteins have been detected also in biological samples, as the likely product of the reaction of L-Met with reactive oxygen species resulting from the aerobic metabolism. Notably, L-Met can be also oxidized by NADPH-dependent flavin monooxygenase enzymes in humans and rabbits, with different stereoselectivity depending on the enzyme isoforms [11]. The reaction occurs also if L-Met has the carboxyl group engaged in a peptide bond, such as in the dipeptides L-Met-L-Val and L-Met-L-Phe, but not if its amino group is blocked, such as in L-Phe-L-Met, Gly-Gly-L-Met and Nacetyl-L-Met [11]. On the other hand, the ability of this class of

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enzymes of stereoselectively oxidising L-Met residues in actin has been recently demonstrated, and it appears that this reaction is implicated in the regulation of macrophage function [12,13]. Significant levels of L-Met-(O) were first detected in the 1970s, in bovine renal glomerulus and lens [14], in human crystalline lens proteins during development of senile nuclear cataract [15], and in the proteins released from granulocytes activated by different agonists of the respiratory burst [16,17]. Several are the proteins that are inactivated after oxidation of some of their L-Met residues, and these include enzymes, protease inhibitors, hormones and chemotactic factors [18]. Already in the 1960s, evidence was obtained that proteins such as phosphoglucomutase [19], chymotrypsin [19] and the pituitary adrenocorticotropin [20] were inhibited as the consequence of L-Met oxidation. Remarkable is the inactivation of the serum protein a1-proteinase inhibitor (a1-antitrypsin), resulting from the oxidation of a critical L-Met residue in its active site [21,22]. Because cigarette smoke was shown to promote oxidation and inactivation of a1-antitrypsin, with loss of inhibition of serum elastase, this oxidative process has been implied in the onset of emphysema [18]. In addition to the loss of biological properties that many proteins undergo upon oxidation of one or more of their L-Met residues to the sulfoxide, also free L-Met-(O) negatively interferes with biosynthetic processes, being unable to combine with adenosine to form the methylating agent S-adenosylmethionine, which operates through the transfer of the methyl group bound to the sulphur atom in L-Met [23], or to be inserted into polypeptides during protein synthesis, because the methionyl-tRNA synthetase does not recognise L-Met-(O) [24], also indicating that L-Met oxidation in proteins is exclusively a posttranslational modification. The presence of the more hydrophilic L-Met-(O) in a position where L-Met should be, may alter the chemical-physical properties of the proteins, as in the case, for example, of the human transmembrane protein glycophorin A, in which the oxidation of three L-Met residues (out of a total of 137 amino acids) causes a change in the mobility of the protein in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [25]. L-Met sulfone, whose formation from L-Met would require very strong prooxidant conditions that are not usually compatible with the cellular environment under physiological conditions, has rarely been detected in vivo. Exceptions are a protein fraction from human cataractous lenses [26], and the catalase of the bacterium Proteus mirabilis, where a residue of L-Met sulfone is located in the active site, probably with the function of preventing the access to non-natural substrates and inhibitors [27]. The first evidence of an enzymatic activity that catalysed the reduction of L-Met-(O) was obtained in the late 1930s, when it was shown that the growth of rats was not changed if the L-Met in their diet was replaced with L-Met-(O) [28], whereas the growth was suppressed when L-Met was replaced by L-Met sulfone [29]. This suggested that either L-Met-(O) (but

Discovery of Methionine Sulfoxide Reductase Enzymes

not the sulfone) could perform the functions of L-Met in the biosynthetic pathways, or that there existed a mechanism that converted L-Met-(O) back to L-Met. Further evidence was obtained that extracts from Escherichia coli [30–32], yeast [33], plants [34], and rat tissues [35] had the ability to reduce free L-Met-(O) thanks to the activity of a NADPH-dependent multienzymatic system, of which it was not possible to clarify the nature. In 1970, two of the proteins involved in the system were identified as thioredoxin (Trx) and thioredoxin reductase (TrxR) [36], while a third enzyme was identified and purified from E. coli only in 1980 [37], and shown to be able to reduce free L-Met-(O). It was also possible to elucidate that the role of Trx/TrxR was that of transferring the electrons from NADPH to the enzyme that catalyses the reduction of L-Met-(O). This observation was later confirmed in experiments with mutant E. coli that expressed inactive Trx forms. These bacteria did not grow in a medium containing L-Met-(O) in place of L-Met when the normal L-Met-generating pathway was blocked, indicating a failure in the mechanism of reduction of L-Met-(O), and that Trx is the only efficient electron-donor involved in this process [38]. Dithiothreitol (DTT) could substitute as the electron donor in the reaction in vitro, but other thiol reagents, such as b-mercaptoethanol and glutathione, could not [37]. Within the study on E. coli it was also clarified that this enzymatic system was unable to reduce the L-Met sulfone [32], whose formation occurs through what should be considered an irreversible reaction, in vivo. The earliest indication of an enzymatic activity capable of reducing residues of L-Met-(O) inserted into proteins, was obtained during the study of the E. coli ribosomal protein L12, which loses its biological function upon oxidation of selected L-Met residues by hydrogen peroxide. It was shown that extracts of E. coli were able to restore the active form of L12 [39]. The enzyme had been partially purified, and showed activity towards L-Met-(O) bound to L-12, and also little activity against free L-Met-(O) [39]. The same enzyme, partially purified from E. coli, was also able to reduce the L-Met-(O) in the active site of canine a1-proteinase inhibitor, and to restore its inhibitory activity toward elastase [40]. This is the first description of restoration of activity to a mammalian protein after reduction by a Msr. Conversely, the enzyme purified to homogeneity in 1980 and capable of reducing free L-Met-(O) [37], did not display the ability of reducing L-Met-(O) residues in L12. The enzyme capable of reducing protein-bound L-Met(O) was then purified to homogeneity from E. coli (and also partially purified from human leukocytes),* and showed its inability in reducing free L-Met-(O) [41]. For these reasons, it was called peptide L-Met-(O) reductase or L-Met-(O) peptide reductase [42], and it was often indicated as pMsr. It should be emphasized that a further study, performed by means of cloning techniques (see below), re-established that this enzyme

*A more recent work has shown that almost all Msr activity in samples of leukocytes is contributed by neutrophil granulocytes [97].

is also active on free L-Met-(O) [43]. The enzyme that reduced free L-Met-(O) [37], was called free L-Met-(O) reductase [44], and is now known by the acronym fSMsr [45]. The free L-Met(O) reductase activity, also that from other cell types, was sometimes indicated as fMsr [46]. It should be stressed that the original classification (fMsr and pMsr) is now considered in less stringent terms, because Msr forms of different organisms reduce L-Met-(O), free or inserted into proteins, with various degrees of specificity. Since the vast majority of cellular L-Met is bound to proteins, and the presence of L-Met-(O) in proteins can profoundly affect their function, mostly in a negative fashion, the interest was oriented predominantly towards research in the field of pMsr. This resulted in the adoption, for the assay of these enzymes, of synthetic substrates where the a-amino group of L-Met-(O) is blocked, in order to simulate the insertion of L-Met-(O) in a polypeptide chain [the derivatization of the a-carboxyl group is also sufficient for mimicking the insertion of L-Met-(O) in a polypeptide chain (see below)]. The first artificial substrate of this type was N-acetyl-L-[3H]Met-(O) [42], with which high levels of Msr activity were first measured in human and bovine lens [47], human neutrophils [42], and some plants, such as spinach, pea and barley [48].

2. The Progresses: Cloning of Msrs and Discovery of Their Stereoselectivity To find out more about the structure and functioning of pMsr, whose re-naming as MsrA was at that time also proposed, a gene was cloned from E. coli in the early 1990s, which was then sequenced and expressed into a recombinant protein [49,50]. The first step towards that result had been the partial sequencing of tryptic peptides obtained from the N-terminal portion of the purified protein from E. coli. By also taking into account the codon usage in E. coli, a non-degenerate sequence of 62 nucleotides was obtained from the N-terminus of the protein, which was then used as a probe for screening a cDNA library of E. coli in phage kgt11 [49,51]. A similar method was applied, in 1996, for the first cloning of a mammalian msrA gene, starting from the N-terminal partial sequence of four tryptic peptides purified from bovine liver, and by screening a cDNA library of bovine adrenal gland. The sequence of a bovine MsrA (bMsrA) was obtained, which turned out to be 61% homologous to the sequence of the E. coli MsrA [43], and was able to reduce both free and protein-bound L-Met-(O). In the same year it was observed that the N-terminal and the central domains of the Neisseria gonorrhoeae PilB protein, so-named because of its involvement in the formation of type IV pilus [52], were homologous, respectively, to Trx and MsrA [53]. Furthermore, the central domain was also enzymatically active towards an equimolar mixture of L-Met-R-(O) and L-MetS-(O) [53], whereas the C-terminal region was not investigated for its function at the time of the characterization of this enzyme. The further sequencing of msrA genes in other

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species was based on the screening of the corresponding cDNA libraries with probes that were constructed only on the basis of the sequence of the MsrAs that were already known (E. coli and bovine). This process led, in 1999, to the sequencing and expression of the first human MsrA (hMsrA), whose homology with bMsrA was 88% [54]. It is interesting to note how this modus operandi of cloning on the basis of homology to a sequence obtained in (a) single episode(s) of purification, resulted in the unwanted narrowing of the field of investigation, leading to the discovery of Msr forms that subsequently proved to be only specific for the reduction of L-Met-S-(O). Furthermore, pMsr of E. coli, and the enzymes that were later purified, were named MsrA† [55], well before the existence of another group of enzymes, those responsible for the reduction of L-Met-R-(O), that were called MsrB [56], was recognized. The first hint to a possible stereospecific character of pMsr was obtained in our lab, where the pMsr activity that was known at that time to be expressed at high levels in human granulocytes revealed to preferentially reduce L-Met-R-(O) [57]. The fact that crude extracts of granulocytes were used partially limited the impact of this discovrery, that was only retrospectively proven to be correct (see below). This observation had been made possible by the use, for the first time in the literature, of the optically pure diastereoisomers of L-Met(O),‡ conjugated via their a-amino group with the chromophore 4-(dimethylamino)azobenzene-4-sulfonate (DABS), used to monitor and quantify the products and the reagents of the enzymatic reaction, after separation by reverse-phase high performance liquid chromatography (rp-HPLC; see below). Only in 1999 results were presented, which demonstrated that recombinant bovine MsrA acted stereospecifically on L-Met-S(O), either free or when present in oxidized calmodulin [58]. These results were obviously at variance with our own previously published data, which showed reduction of L-Met-R-(O) by human granulocytes. This discrepancy, however, was important for bringing the attention to this stereochemistry aspect, and for explicitly suggesting, for the first time, the possible existence of other, still unknown forms of Msr with different substrate stereospecificity. In 1999 two new proteins were identified in mammals, through in silico studies, that were homologous to the Cterminal domain of PilB of Neisseria gonorrhoeae, but whose biological function was initially ignored. One of these proteins,

†

The acronym MsrA was originally used to describe the only peptide methionine sulfoxide reductase from E. coli and it was coined because the existence of other methionine sulfoxide reductases had already been hypothesized. ‡ In the cited work, the two diastereoisomers of L-Met-(O) were not named S or R according to the absolute configuration around their sulphur atom, but with the then widely used nomenclature based on their ability to rotate the plane of linearly polarized light to the left or to the right. According to this, the dextrorotatory isomer, L-Met-d-(O) corresponds to L-Met-S-(O), while the levorotatory isomer, L-Met-l-(O), corresponds to L-Met-R-(O).

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called CBS-1 because the gene was identified in a cDNA library of human ciliary body, was discovered within a study focused on the identification and characterization of tissue-specific genes differentially expressed in this ocular tissue [59]. The second protein was identified simultaneously by two independent groups, and was named SelR [60] or SelX [61] because it was shown to be a selenoprotein, containing a residue of Sec. The identification had made possible, in the context of a study of the human selenoproteome, by the development of novel computational programs used to scan genomic data bases in order to recognize those potential secondary structure elements in the mRNA molecule, the Sec insertion sequence (SECIS) elements, that must be present, 30 from the stop codon, in the genes encoding for eukaryotic selenoproteins. The SECIS elements allow, thanks to the existence of a specific elongation factor, the translational re-coding of what would otherwise be read as a stop codon as the codon for Sec insertion. Because Sec is encoded by a stop codon which is translationallyrecoded, also thanks to sequences that lie outside of the open reading frame, the traditional computational tools could not be used to predict the presence of selenoproteins in a given genome. In 2001 two new additional proteins that shared 50% of homology with the C-terminal region of PilB were identified in E. coli [56] and Staphylococcus aureus [62], and named, respectively, YeaA and PilB homolog. Most importantly, the two proteins exhibited Msr activity but, simply because they were homolog to the C-terminal domain of PilB, whose function had not been recognized yet, and not to the central domain of PilB, which was common to other MsrA proteins, they were named MsrB. It should be added that for both proteins the substrate stereospecificity was not investigated because a mixture of the two diastereoisomers of L-Met-(O) was used as the substrate in the enzymatic assay, a common procedure in those years [56]. Eventually, in 2002 the C-terminal region of PilB was cloned and expressed, revealing, by direct enzymatic analysis with the pure diastereoisomer, stereospecificity towards L-MetR-(O) [63,64]. The same stereospecificity was simultaneously reported for mouse and Drosophila melanogaster SelR [65,66], for the PilB homolog expressed by Staphylococcus aureus [66], for human CBS-1 and for YeaA of E. coli [63]. Shortly thereafter, through studies of sequence homology with the Cterminal domain of PilB, various MsrBs were identified in E. coli [45] and mammals, including humans [67,68]. In the same years, tandem Msr domains similar to that of Neisseria gonorrhoeae were also found in Neisseria meningitidis, Helicobacter pylori, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus gordonii [63], and in the hyperthermophilic Archaeobacterium Thermococcus kodakaraensis [69]. An exception to the arrangement of the two catalytic domains was shown to occur in the Treponema pallidum PilB homolog, where the MsrB domain is situated in the N-terminal, not in the C-terminal of the protein, and in the plant Arabidopsis thaliana homolog, which lacks the MsrA domain, and in which

Discovery of Methionine Sulfoxide Reductase Enzymes

two MsrB domains are present instead [63]. More recently, in the alga Gracilaria gracilis a fusion protein has been found that consists of a MsrA in the C-terminal domain and two functional glutaredoxins in the N-terminal domain. This shows that the glutathione/glutaredoxin system is also able to act as the reducing agent for MsrA with an efficiency comparable to that of the Trx/TrxR/NADPH system [70]. Thus, the identification of MsrBs as the family of Msr stereospecific for L-Met-R-(O), followed a rather intricate pathway, and was possible largely because of the existence of a natural chimera of Msr domains with opposite stereospecificity and unrelated evolutionary lineage, in the form of the Neisseria gonorrhoeae PilB. The catalytic activity of the PilB central MsrA-type domain permitted to originally classify PilB as a Msr, while the homology of its C-terminal domain with other proteins permitted to fish them out through in silico studies and to discover their catalytic activity, which only later turned out to be directed towards the R diastereoisomer of L-Met-(O), and to classify them as MsrBs on the basis of the stereoselectivity criterion. Thanks to genomic studies and cloning techniques, the research around Msrs has received a remarkable propulsion in the last two decades, showing the ubiquitous distribution of these enzymes, from prokaryotes to eukaryotes, including plants and some archaea [71–73], and the expression of many isoforms in the same organism, such as in the plant Arabidopsis thaliana, where five MsrAs and nine MsrBs have been found [71], and in E. coli, in which, over the years, the presence of a total of six Msrs has been established. Three of them have activity towards L-Met-S-(O): MsrA (originally called pMsr), that reduces both free and protein-bound L-Met-S-(O) [39,41,43], MsrA1, only active towards peptide-bound L-Met-S(O) [74], and fSMsr, that catalyses the reduction of free L-MetS-(O) [32,37]. The two forms that reduce L-Met-R-(O) are MsrB (originally named YeaA), prevalently active on the proteinbound sulfoxide (56), and fRMsr, active on the free amino acid only [45,75].§ The sixth member is a membrane-bound enzyme called mem-R,S-Msr, that catalyses the reduction of both diastereoisomers, either free or inserted into peptides [74]. The latter enzymatic activity, still poorly studied, could be due to two different membrane proteins or by a protein equipped with both MsrA and MsrB domains. Several forms of MsrA and MsrB are also expressed in mammals, including humans. In addition to the well-known MsrA, that was purified and cloned from bovine tissues in the 1990s [43] and reduces both free and protein-bound L-Met-(O) [76], more recent in silico studies have shown that different variants of MsrA exist [77]. One of these, indicated as MsrA(S), is originated from the alternative splicing of the mRNA for MsrA, and is characterized by a shorter sequence and an §

This enzyme is also reported as MsrC in EcoGene database, on the basis of the recent renaming of the corresponding gene from yebR to msrC. Other databases, such as Swiss Prot and UniProt, use the traditional and more rigorous short name fRMsr.

enzymatic activity three times lower than that of the nontruncated form [77]. Other forms generated by alternative splicing were also detected, but were enzymatically inactive [77]. It has been hypothesized that the alternative splicing, as a common mechanism that contributes to vary the subcellular distribution of proteins, allows the targeting of MsrAs for the repair of oxidized L-Met residues in proteins belonging to different compartments [77]. Furthermore, it has been shown that, in mammals, MsrA also exist in a myristoylated form, localized solely in the cytosol, but the role of this lipidation has not yet been ascertained [78]. With regard to mammalian MsrBs, their classification was for some time rather confusing and easily lending itself to misinterpretation. In fact, at the end of 2003 the gene for a new form of human MsrB was identified, which was called MsrB2, while CBS-1, which had been discovered earlier, was renamed MsrB1 [67]. At the same time, however, another group identified the same gene of the new MsrB protein in the human and mouse genomes, whose primary transcript mRNA was subjected to alternative splicing, generating two different proteins that were named MsrB3A and MsrB3B. With the new finding, these authors renamed the other two previously discovered MsrBs in a different way: SelX (SelR) became MsrB1, while CBS-1 was called MsrB2 [68,79]. After a few years, during which both systems of nomenclature coexisted [80,81], consensus was reached on the latter form of classification [68]. Among mammalian MsrBs, significant is the presence of MsrB1, one of the only twenty-five selenoproteins that have been identified to the present day in mammals,¶ and the only member of the MsrB family that contains a Sec. MsrB1 owes to the presence of this Sec in its catalytic site the approximately four times higher specific activity for a given substrate with respect to other members of the MsrB family [68,82]. Only certain bacteria, algae and invertebrates express forms of MsrA that are equipped with a catalytic residue of Sec, and have an enzymatic activity ten to fifty times higher than that of the Cys-containing MsrA [83,84]. In the mechanism of the reaction, which is essentially the same for MsrA and MsrB forms, despite the structural differences existing in their fold, one catalytic Cys (or Sec) intervenes in the first step of catalysis, that contemplates the nucleophilic attack of a thiolate (or selenolate) group to the sulphur atom of L-Met-(O), with the oxidation of the thiolate (or selenolate) to sulfenic (or

¶

To date, 25 different selenoproteins have been identified in the human genome [168], some of which can be grouped in families with similar function: glutathione peroxidase (GPx; five genes), thioredoxin-reductase (TrxR; three genes), iodothyronine deiodinase (DIO; three genes) and selenophosphate synthetase (SPS2). The remaining proteins have been annotated according to the following alphabetical order: Sep15, SelH, SelI, SelK, SelM, SelN, SelO, SelP, SelR, SelS, SelT, SelV, SelW. The few selenoproteins that have been functionally characterised in detail are oxidoreductase enzymes that contain a catalytically active Sec: GPx, TrxR, DIO, SPS2, SelP and SelR. They are all involved in the regulation of the redox homeostasis [169].

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selenenic) acid and the release of L-Met [85]. The higher reactivity of Sec over that of Cys is due to the greater nucleophilicity of selenolate compared to thiolate, that increases the rate of the reaction by promoting the nucleophilic attack [86]. In some Msrs, a special role is played by certain additional Cys residues, which bring back the oxidized active site to the reduced state after one round of catalysis, thus recycling the enzyme. These recycling Cys residues perform their thiol/disulfide exchange by transferring the electrons from the Trx/TrxR/ NADPH system to the oxidized catalytic centre. The number of recycling cysteines depends on the Msr isoform: in mammalian Msrs there are two recycling cysteines for MsrA, one for MsrB1, and none for MsrB2 and MsrB3 [85]. For a schematic explanation, see Fig. 2, and for a detailed review about the catalytic mechanism of Msr enzymes, see [87]. A novel 5-kDa selenoprotein was detected in mouse. It is not generated by alternative translation initiation, but is probably derived from post-translational cleavage of full-length MsrB1 (14 kDa). It corresponds to the C-terminal domain of MsrB1, where the recycling Cys is deleted, and it seems to have no catalytic activity. The biological role of such protein has not yet been investigated [88,89].

3. Methods for the Measurement of Msr Activity The interest that grew around Msrs beginning in the 1980s led to the development of numerous methods for assaying their enzymatic activity towards both free- or protein-bound L-Met(O). As it should be normally expected, a given Msr form does not show the same catalytic activity for all the artificial derivatives of L-Met-(O) and for all the residues of L-Met-(O) inserted into proteins, because differences in the chemical structure of the substrate could affect its affinity for the enzyme, by introducing disturbing factors in the form of electronic and/or steric effects. This aspect has not been well investigated, but it has been found that the reduction of L-Met-(O) depends on the position occupied by the residue within the protein. In fact, yeast MsrA and MsrB were more efficient in reducing L-Met(O) in unfolded than in folded proteins, and this phenomenon seems to be due to better access to the substrate in denatured proteins by Msrs [90]. For the same reason, probably, the LMet-oxidized apolipoprotein C-II is a more suitable substrate for human MsrA and MsrB2 when in native, monomeric form than when it has undergone conversion to the fibrillar amyloid structure [91]. Furthermore, in activated granulocytes, it has been shown that newly synthesized proteins contain more LMet-(O) that the other proteins, suggesting that L-Met-(O) in nascent polypeptide chains could be more susceptible to oxidation than those located within the tertiary structure of a mature protein [16]. The first method to assay the pMsr activity in E. coli [39] was based on the observation that the enzyme L12transacetylase is capable of transferring an acetyl group to the

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FIG 2

Schematic representation proposed for the catalytic mechanism of Msr enzymes. (A) Mechanism for MsrAs equipped with a catalytic cysteine (referred to as A) and two recycling cysteines (B and C). Initially the enzyme contains all three cysteines in the reduced form (1), the first step is the oxidation of cysteine (Cys) A to sulfenic acid and the reduction of one molecule of L-Met-(O) (2). Then, the formation of a disulfide bond between Cys A and Cys B occurs (3), followed by the reduction of Cys A to the thiolate and the formation of a disulfide bond between Cys B and Cys C (4). At this point, the thioredoxin (Trx)/thioredoxin reductase (TrxR)/NADPH system could reduce the disulfide bond and restore the enzyme in the fully reduced form (1). However, experimental evidence has shown that the actual reaction mechanism proceeds from (4) with an additional oxidation of Cys A to sulfenic acid and the reduction of another molecule of L-Met-(O) (5), with the further reduction of the disulfide bond between Cys B and Cys C by Trx (6). At this point, the cycle begins again from (3). The presence of two recycling cysteines induces two successive reductase steps, with the formation of two molecules of L-Met and the release of two molecules of water derived from the reduction of sulfenic acid. (B) Mechanism for Msrs (MsrAs and MsrBs) equipped with only one recycling cysteine: at (3) the disulfide bond between Cys A and Cys B is reduced by Trx, and one molecule of L-Met is produced in every cycle. (C) Mechanism for Msrs (MsrAs and MsrBs) without recycling cysteines: the sulfenic acid is directly reduced by Trx, but other reductants, such as thioneine, glutaredoxins and some selenocompounds could be involved [87]. A stereospecific oxidase activity has been observed in MsrAs equipped with two recycling cysteines, in which the sulfenic acid intermediate obtained in the second reductase step (5) can revert to the thiolate form with the oxidation of a molecule of L-Met [87,144].

N-terminal serine of the E. coli ribosomal protein L12 (thus forming L7) only if the three L-Met residues of L12 are in the reduced form [92]. The oxidation of L-Met residues in L12, besides inhibiting its acetylation, also prevents the incorporation of L12 into ribosomes, due to the protein’s inability to assume the functionally active, dimeric L7 form [92]. The method required the preparation of the substrate as the oxidized form of L12 (a relatively easy task owing to the absence

Discovery of Methionine Sulfoxide Reductase Enzymes

in L12 of oxidation-prone amino acids other than L-Met), which was then incubated with the cell extract to be tested. As a source of reducing equivalents, DTT was used. The presence of Msr activity and, therefore, the reduction of L-Met-(O) residues in L12 back to L-Met, was measured by assessing the acetylation of L12 by L12-transacetylase in the presence of 3Hlabeled acetate. The level of incorporation of radioactivity thus reflected the degree of reduction of L-Met-(O) in L12. With this method, Msr activity was originally detected, in addition to E. coli, also in Euglena gracilis, Tetrahymena pyriformis, spinach, HeLa cells, and in many rat organs [39]. It was also demonstrated that the enzymatic activity present in E. coli was able to reduce L-Met-(O) not only when the amino acid occupied an internal position in the polypeptide chain, but also when it represented the C-terminal residue, as in the case of L-Met-(O)-enkephalin (the oxidized form of the endogenous opioid peptide L-Met-enkephalin) in which the Cterminal L-Met-(O) was reduced by pMsr [39]. This experimental evidence provided the rationale for the definition of the sufficient condition for a potential pMsr substrate as that of having the amino group of L-Met-(O) engaged in a carbamidic bond. This opened up the possibility of developing synthetic substrates in which the L-Met-(O) amino group is blocked by different various compounds. The first synthetic substrate that was developed according to this rule was N-acetyl-L-[3H]Met(O), in which the insertion in a polypeptide chain was mimicked by the acetylation of the amine moiety. The labelling with 3H on the methyl group of L-Met-(O) allowed to detect enzymatic activity by measuring the level of radioactivity recovered in the product of the reaction, N-acetyl-L-[3H]Met, after separation of the latter from the substrate and reactants by extraction of the reaction mixture with ethyl acetate [42]. A variant of this method was also proposed, that was based on the use of the chemotactic peptide [3H]N-formyl-L-Met-(O)-LLeu-L-Phe as the substrate [93]. Also in this case, the product was separated from the reagent by means of extraction with ethyl acetate. Another assay that was proposed for the determination of Msr activity in chloroplasts of Pisum sativum was based on the reaction of L-Met-(O) with the fluorochrome N-(9-fluorenylmethoxycarbonyl) chloride (FMOC) that binds to the amino group with formation of a carbamidic bond [FMOC-L-Met-(O)]. The product of the enzymatic reaction (FMOC-L-Met) is separated from FMOC-L-Met-(O) by rp-HPLC and quantified with a fluorimetric detector [94]. A non-natural Msr substrate that received greater approval, and that is at the basis of what seems to be now the most widely used analytical method, is 4(dimethylamino)azobenzene-4-sulfonyl-L-Met-(O) [DABS-L-Met(O)] [57], obtained by functionalization of the amino group of LMet-(O) with DABS chloride, with the formation of a sulfonamide bond [95]. In the original proposal of the method, care was taken to choose among suitable reducing compounds, and dithioerythritol (DTE) was preferred over the most commonly used diastereoisomer DTT, to minimize an unwanted reaction that was observed to occur between the dithiol and the DABS

moiety, and that probably resulted in the formation of the corresponding hydrazo moiety of the dye, with significant loss of the absorbance at 436 nm [57]. By using DTE, and by limiting reaction time and temperature to the suggested values, the unwanted, bleaching reactions were almost completely eliminated. The advantages of the DABS method consist in the ease and reproducibility of the derivatization procedure, the high water solubility of the azo dye (the hydrolytic product of DABS chloride is nothing else than the familiar pH indicator methyl orange) and its derivatives, and the straightforward and trouble-less HPLC-based quantification of the reaction product with an absorbance detector. During the development of the DABS method for assaying Msr activity, a parallel investigation was undertaken in our lab to verify whether the Msr activities that were known at the time could act stereospecifically towards the two diastereoisomers of L-Met-(O). Being aware of the existence of two LMet-(O) isomers, that were also detected with the then familiar and diffuse Stein & Moore automated amino acid analyser [96], we speculated that stereospecific chemistries could have evolved in nature to selectively deal with the two stable sulfoxides. Therefore, we set out to resolve the racemic mixture of LMet-RS-(O) [or L-Met-dl-(O), as we knew it at the time] by salification with picric acid and fractional crystallization of the picrate salts [8]. The DABS derivatives of the two optically pure diastereoisomers were prepared and tested with crude extracts of human granulocytes, which were known at the time to express high levels of pMsr activity. Reagents and products of the reaction were separated by rp-HPLC. As already mentioned, it was observed that granulocyte extracts preferentially reduced DABS-L-Met-R-(O) to DABS-L-Met [57]. In retrospect, the almost exclusive reduction of L-Met-R-(O) that we observed using granulocyte extracts is easily explained by the expression in this cell type of high levels of MsrB1, the only known selenoenzyme among eukaryotic Msrs. Not only is MsrB1 much more active than the corresponding Cyscontaining enzyme, but it happens to be selectively expressed at much higher levels with respect to other Msrs in this cell type, as judged by mRNA quantification [97]. All these coincidences made it easier the discovery of the stereospecificity of pMsr [57,58]. Methionine-oxidized calmodulin has been proposed in various methods developed for the analysis of MsrA [58,98], and it proved to be valid also for the assay of MsrB activity [56]. The first method [98] was based on the loss, by calmodulin, of the ability to activate the plasma membrane-Ca21-ATPase when some of the nine L-Met residues in calmodulin itself were oxidized to the sulfoxide, and on the ability of Msrs to reactivate oxidized calmodulin. The Msr activity levels were assayed by quantifying the amount of phosphate released by active Ca21ATPase, and, by mass spectrometry, the levels of L-Met oxidation in calmodulin [98]. The second calmodulin-based method [58] relied on the tryptic digestion of oxidized calmodulin after incubation with MsrA, the separation of tryptic peptides by rpHPLC, the collection of the fragments that contained L-Met-(O)

141

BioFactors

FIG 3

Structures of some synthetic substrates used to assay the catalytic activity of Msr enzymes: (1) N-acetyl-L-Met-(O) (with the methyl group of the side chain labelled with 3H); (2) N-(9-fluorenyl-methoxycarbonyl)-L-Met-(O) [FMOC-L-Met-(O)]; (3) 4-(dimethylamino)azobenzene-4-sulfony-L-Met-(O) [DABS- L-Met(O)].

(identified by mass spectrometry) and the further digestion by carboxypeptidase to obtain the single amino acids. Finally, the detection of the diastereoisomers of L-Met-(O) was performed by pre-column derivatization with o-phthalaldehyde and rpHPLC analysis with fluorimetric detection. With this method, the stereoselectivity of MsrA towards protein-bound L-Met-S(O) was described for the first time [58]. A synthetic peptide (KIFMK) containing L-Met-(O) was also adopted for the detection of Msr activity, using mass spectrometry for the analysis of the oxidized and reduced peptides [99]. In the assays based on oxidized calmodulin and on the synthetic peptide, DTT or Trx/TrxR/NADPH were used as reducing agents. When the natural electron-donor was employed, the presence of Msr activity could also be detected by the decrease in absorbance at 340 nm, corresponding to the oxidation of NADPH [56,99]. Assuming that the oxidation of L-Met in calmodulin generated a mixture of the two diastereoisomers of L-Met-(O), the fact that the full repair of calmodulin required the presence of both MsrA and MsrB suggested their different stereoselectivity [56]. Direct demonstration of Msr stereospecificity, however, was provided only one year later, when separate L-Met-R-(O) and L-Met-S-(O) were used as substrates. For this scope, different approaches have been adopted, based on free L-Met-(O) [nitroprusside method and radiolabelled L-Met-(O), see below] and functionalized L-Met-(O), to mimic the insertion in a polypeptide chain. In addition to the classic DABS-L-Met-(O) [65,66], also N-acetyl-L-Met-(O), L-Met-(O)-NHMe and N-acetylL-Met-(O)-NHMe were used [64]. The latter are L-Met-(O) derivatives in which the a-amino or the a-carboxy groups (or both) are engaged in amide bonds (in the first case the ligand is an acetyl group, in the second a methyl-amine group). The analysis was performed by the spectrophotometric measurement of NADPH oxidation [64]. Among these derivatives, N-acetyl-LMet-(O)-NHMe was the most efficient substrate for both MsrA and MsrB domains of PilB, and this indicated that, although a single functionalization of the a-amino group (as already demonstrated) or of the a-carboxy group (as reported in the cited paper for the first time) is sufficient to allow the reduction of L-Met-(O) by Msrs specific for protein-bound L-Met-(O), the

142

double blockage mimicked more satisfactorily the insertion of L-Met-(O) in a polypeptide chain. In the same years, the pentapeptide L-Tyr-D-Ala-Gly-L-Phe-D-Met-(O) [D-Met-(O)-enkephalin] had been used as a substrate to assay the Msr activity of membrane vesicle preparations from E. coli (about the reduction of D-Met-(O) by Msrs, see below). The procedure, that included the separation by rp-HPLC of the reaction product (D-Metenkephalin) from the reactants [the two diastereoisomers of DMet-(O)-enkephalin], proved that E. coli is endowed with a membrane-bound Msr activity (named mem-R,S-Msr) that is able to reduce both diastereoisomers of D-Met-(O) [and consequently both diastereoisomers of L-Met-(O)] inserted into polypeptides [74]. More recently, some L-Met-rich proteins, after appropriate oxidation, have been proposed as substrates for Msr enzymes. The semi-quantitative detection of Msr activity can be performed by SDS-PAGE analysis, by comparing the intensity of the electrophoretic bands of the oxidized and reduced forms of the protein, that show a difference in electrophoretic mobility, or by immunological methods such as Western blotting and ELISA, using an antibody specific towards these oxidized proteins [100,101]. Finally, several methods based on capillary electrophoresis have been recently proposed, that allow faster analysis of L-Met and of the two diastereoisomers of L-Met-(O) functionalised with DABS [102,103], FMOC [104], or inserted in modified pentapeptides [105,106]. In Fig. 3 the chemical structures of some derivatives of L-Met-(O) used for the assay of Msr activity are reported. Numerous have also been the methods employed for the analysis of Msr activity with free L-Met-(O) as the substrate. They were based on the following criteria: the measure of the NADPH oxidation by monitoring the decrease of absorbance of the reaction mixture at 340 nm [33]; the radioactive labelling of L-Met-(O) with 14C, followed by the separation of substrate and product by paper chromatography and the analysis of radioactivity by liquid scintillation counter [34]; the nitroprusside method, with absorbance reading at 540 nm [32,63], and the enzymatic amino acylation of tRNAMet with L-[35S]Met, in which the amount of formed L-Met-tRNA resulted to be proportional to the concentration of L-Met produced in the sample by enzymatic reduction of free L-Met-(O) [37]. An alternative procedure is the pre-column derivatization with o-phthalaldehyde (which binds to the a-carboxy group of amino acids) and rpHPLC analysis of residual L-Met-(O) after incubation with the enzyme, monitoring the elution by fluorescence detection, essentially as for the method with oxidized calmodulin as the substrate (see above) [58]. By means of this technique, which allows the separation of the two diastereoisomers of L-Met-(O), the stereoselectivity of bovine MsrA for free L-Met-S-(O) was described [58]. Another radioactivity-based method was adopted during the studies concerning the catalytic mechanism of MsrA [46] and the discovery of the stereospecificity of MsrBs [66]. L-[3H]Met-(O) or L-[35S]Met-(O), after incubation with Msr and DTT, were analyzed by thin-layer chromatography (TLC) on a silica gel plate. After ninhydrin staining, the

Discovery of Methionine Sulfoxide Reductase Enzymes

spot that corresponded to L-Met was extracted by water, and the radioactivity was measured. Furthermore, a qualitative method used to detect Msr activity towards free L-Met-(O) was based on the separation of L-Met and L-Met-(O) on TLC silica plate and their staining with ninhydrin or iodine vapours [45,68]. Finally, it has also been proposed to monitor the enzymatic reduction of free L-Met-(O) by comparison of the nuclear magnetic resonance (NMR) signal intensities of the methyl moiety in L-Met and L-Met-(O) [107], or by measuring the decrease in DTT concentration in the reaction mixture with the Ellman’s spectrophotometric assay [108]. As illustrated in this chapter, a number of methods have been developed over the last 30 years for the detection of Msr activity toward both free and protein-bound L-Met-(O). For protein-bound L-Met-(O), a variety of L-Met-(O) derivatives have been proposed as substrates, where L-Met-(O) is suitably modified to mimic its insertion in the polypeptide chain. The analysis of Msr activity is based on the separation of the product from the substrates by means of different techniques such as differential solvent extraction, rp-HPLC, capillary electrophoresis, followed by detection with spectrophotometric, spectrofluorimetric or radioactivity counting techniques. Synthetic or natural small peptides and specific proteins, with the L-Met residues properly oxidized to L-Met-(O), have also been used as Msr substrates, and their reduction measured by several spectroscopic methods, such as NMR and mass spectrometry, or by assessing the restoration of their biological properties. Although all of these methods have proved successful and valid for the measurement of Msr activity, both in cell extracts or tissues, and for the determination of the catalytic properties of the pure enzymes, specific pro et contra are inherent to each of the methods, so that their absolute ranking is not possible. In Table 1 a summary is presented of the various methods with comments on their major advantages/disadvantages.

4. Other Msr Substrates The possibility for Msrs to catalyse the reduction of the sulfoxide group in molecules that are not structurally related to LMet has been taken into account quite early. In 1996 it was observed that bovine MsrA displays a rather broad substrate specificity and can catalyse, in vitro, the reduction of a wide range of exogenous molecules containing a methyl-sulfoxide group, such as tetramethylene sulfoxide, dimethyl sulfoxide, and methyl p-tolyl sulfoxide (the enantiomer of S configuration) [43]. Methyl phenyl sulfoxide and other methyl phenyl sulfoxides substituted in the para position of the aromatic ring (with, for example, -NO2, -OCH3, -Br, -Cl) are reduced by bovine MsrA, with an elevated enantiospecificity towards the enantiomer of S configuration, yielding enantiomeric excesses higher than 90%. No correlations between electronic effects of the substituents according to the Hammett equation [109] and the kinetic activity of MsrA were observed [110]. D-methionine sulfoxide is also a substrate for bovine MsrA, which shows an activity comparable to that displayed with the

natural substrate L-Met-(O) [43], indicating that the configuration D or L of the a-carbon atom in L-Met-(O) does not affect the recognition of the substrate by this enzyme, at least for MsrA and also for mem-R,S-Msr from E. coli (see previous chapter). It is evident from the reported data that MsrA shows specificity towards substrates that contain a methyl-sulfoxide group, for which it also displays absolute stereospecificity for the S configuration of the asymmetric centre, while the replacement of the methyl group with a group of greater steric hindrance tends to inhibit the activity. In fact, diphenyl sulfoxide [43] and S-carboxymethyl cysteine sulfoxide (the sulfoxide of S-carboxymethyl cysteine, or carbocysteine, a mucoregulatory drug) [111] are not reduced, whereas L-ethionine sulfoxide (an homologue of L-Met that contains an ethyl group in place of the methyl) [43] and ethyl phenyl sulfoxide [110] are reduced, albeit with lower efficiency with respect to compounds that contain a methyl group as one of the substituents on the sulfoxides sulphur. After these earlier investigations, the research of nonnatural substrates for Msrs has been focused on bioactive compounds equipped with a methyl-sulfoxide moiety. The nonsteroid anti-inflammatory drug sulindac (that acts as an inhibitor of cyclooxygenases 1 and 2, and also appears to have anticancer properties by stimulating apoptosis of cancer cells) is available commercially in the inactive form (sulfoxide, as a mixture of two enantiomers), and to exert its pharmacological activity must be reduced to the sulfide form in vivo. Early experimental evidence showed that sulindac is a substrate for aldehyde oxidase in extracts of Guinea pig liver** [112], but more recently it has been shown that also Msrs are capable of catalysing this reaction. The earliest evidence of reduction of sulindac by human MsrA dates back to 1996, when the drug was tested for its ability to act as a competitor in the enzymatic assay with N-acetyl-L-[3H]Met-(O) [43]. Further studies evidenced that MsrA and mem-R,S-Msr from E. coli are able to reduce sulindac (with selectivity for the S and R enantiomer, respectively), while the other Msr isoforms of E. coli are inactive or poorly active [113]. Extracts of calf liver, kidney and brain were also shown to reduce sulindac, with the highest specific activity being displayed by kidney extracts. Inhibition of sulindac reduction was observed if L-Met-S-(O), but not LMet-R-(O), was added to the reaction mixture, indicating an involvement of MsrA in the reaction [113]. A more recent study revealed that mammalian MsrB1, but not MsrB2 or MsrB3, shows some degree (although not precisely quantified) of reducing activity towards the R enantiomer of sulindac [114]. On the basis of the evidence obtained so far, Msrs appear to be largely responsible for the generation of the pharmacologically active form of sulindac within the body.

**Aldehyde oxidase displays sulfoxide reductase activity, for example, towards sulindac but also toward phenothiazine sulfoxide, diphenyl sulfoxide, dibenzyl sulfoxide and biotin sulfoxide methyl ester, but it does not exhibit a significant activity towards L-Met-(O) [112].

143

144 Methods described for analysis of free Msr activity. The substrate is obviously common to all methods. Pros and cons depend on the system adopted for the separation and analysis of free L-Met.

First simplified method; based on radioactive substrate with associated problems; separation of reagents and products by solvent partition is easy but probably inefficient; substrate not commercially available and difficult to prepare. Variant of the N-acetyl-L-[3H]Met-(O) method; ut supra.

A) spectrophotometric measurement of NADPH B) radiolabelling of L-Met-(O) with 14 C (a carbon or methyl group) C) nitroprusside method D) radiolabelling of L-Met-(O) with 35S and analysis of the enzymatic amino acylation of tRNAMet E) pre-column derivatization and HPLC analysis F) NMR G) measurement of residual DTT by Ellman’s assay only L12 with reduced L-Met residues enzymatically incorporates [3H]-acetyl radiolabelling of the methyl group in the side chain

[3H]-labelled substrate. Radioactivity measurement

L-Met-(O)

Oxidized ribosomal protein L12

N-acetyl-L-[3H]Met-(O)

N-formyl-L-Met-(O)-L-Leu-L-Phe

A) Relatively simpler method, yet fluorimetry more difficult and costly than spectrophotometry; substrate not commercially available but easy to prepare. B) Fast analysis and good resolution of the two diastereoisomers of L-Met-(O). A) Probably the most widely adopted substrate, not available commercially but easy to prepare and to analyse. B) See (C) above.

A) HPLC with fluorimetric detection B) capillary electrophoresis

A) HPLC with absorbance detection B) capillary electrophoresis

FMOC-L-Met-(O)

DABS-L-Met-(O)

Interesting for historical reasons; difficult to implement for routine analysis.

Notes

Substrates and analytical methods used for the measurement of Msr activity

Analytical methods

Substrates

TABLE 1

[57,102,103]

[94,104]

[93]

[42]

[39]

[32–34, 37,45,46, 58,68,107,108]

Refs

BioFactors

Discovery of Methionine Sulfoxide Reductase Enzymes

145

Interesting but not yet validated on a large scale.

HPLC with absorbance detection A) SDS-PAGE B) SDS-PAGE 1 Western blotting C) ELISA

D-Met-(O)-enkephalin

L-Met-rich

[100,101]

[74]

[64]

[56,58,98]

[99,105,106]

Refs

DABS, 4-(dimethylamino)azobenzene-4-sulfonate; DTT, dithiothreitol; L-Met, L-methionine; HPLC, high performance liquid chromatography; MS, mass spectroscopy; NMR, nuclear magnetic resonance; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; Trx, thioredoxin; TrxR, thioredoxin reductase

proteins

Simple method but very time consuming, not suitable for routine analysis.

spectrophotometric measurement of NADPH

N-acetyl-L-Met-(O) L-Met-(O)-NHMe N-acetyl-L-Met-(O)-NHMe

All these synthetic substrates, like others, can be used in a system with Trx/TrxR/ NADPH; probably of low sensitivity.

A), B) Of historical interest, but not suitable for routine analysis. C) Requires the full reducing system Trx/TrxR/NADPH; probably of low sensitivity.

A) measurement of the activation of Ca21-ATPase, MS B) tryptic digestion, HPLC, MS, pre-column derivatization, HPLC with fluorimetric detection C) spectrophotometric measurement of NADPH

Oxidized calmodulin

Notes A) Difficult to adopt routinely. B) Requires the full reducing system Trx/TrxR/NADPH; probably of low sensitivity. C) See (B) above.

Analytical methods A) MS B) spectrophotometric measurement of NADPH C) capillary electrophoresis

(Continued)

Short synthetic peptides

Substrates

TABLE 1

BioFactors

FIG 4

Structures of some natural and synthetic sulfoxides that have been shown to be substrates for Msr enzymes: (1) sulindac; (2) mesoridazine; (3) sulmazole; (4) sulforaphane from Cruciferae; (5) triclabendazole sulfoxide; (6) a-5-methylxylofuranosyl sulfoxide (R 5 lipoarabinomannan) from Mycobacterium tuberculosis; The following compounds have been shown not to be substrates for Msrs: (7) methyl sulfoxide adenosine derivatives from Herdmania momus (the isomer with the substituent in position 3 is also not a substrate); (8) S-carboxymethyl cysteine sulfoxide.

In 2005 it was shown that the natural protector against oxidative stress sulforaphane is a substrate for MsrA [115], and more recently it has been demonstrated that this compound, similarly to the neuroleptic mesoridazine, the cardiotonic sulmazole, and the sulphur-oxidized metabolite of the anthelmintic triclabendazole, can be reduced by mouse MsrA (only the S stereoisomer), but not by mouse MsrB1 and MsrB2 [116].†† The in vitro results indicate a possible strong involvement of MsrA in the metabolism of these drugs, and that MsrA, in contrast to MsrBs, has a broader substrate specificity and is able to reduce with high efficiency substrates structurally very different from L-Met-(O), provided that they contain a methyl group as one of the substituents on the sulfoxide sulphur. This phenomenon could be related to the evidence showing that mammals reduce preferentially free L-Met-S-(O) but not free L-Met-R-(O) [117], and that MsrBs, while only partially able to reduce free L-Met-(O), display much higher reducing activity towards L-Met-(O) inserted into polypeptide chains, indicating a strong selectivity for proteinaceous substrates

††

In the same study it was also shown that dimethyl sulfoxide is a substrate for mouse MsrA, but not for mouse MsrBs. In another study it was described that dimethyl sulfoxide is a strong inhibitor of yeast and mammalian MsrAs, and a mild inhibitor for mouse MsrB2 and MsrB3 [170].

146

[68,118]. The low activity of mammalian MsrBs towards free LMet-(O) could be at least partially compensated by an epimerase activity, whose presence has been hypothesized in mice, yeast and E. coli [119–121] and that would be able to convert L-Met-R-(O) to L-Met-S-(O). Among sulfoxides that are present in nature, MsrA is capable of reducing S-methyl cysteine sulfoxide (the oxidized form of S-methyl cysteine, a minor natural amino acid) [122], and the sulphur-oxidized form of the a-5-methylthioxylofuranosyl moiety of lipoarabinomannan, a sugar present in the wall of Mycobacterium tuberculosis, whose function is believed to be that of preserving the microorganism from the attack of macrophages [123]. The oxidized form of this carbohydrate represents the only other known natural substrate for MsrA apart from L-MetS-(O). Conversely, MsrA cannot reduce the methyl sulfoxide adenosine derivatives that have been recently found in the marine solitary ascidian Herdmania momus [124], and also in the mushroom Ganoderma lucidum [125], that, on the basis of their chemical structures, could potentially find application for pharmaceutical purposes as antibacterial, analgesic, sedative, and cardiac depressant drugs. However, the authors did not determine the configuration of the asymmetric sulphur atom [124], and for this reason it cannot be excluded that the configuration was R, and that these compounds could be substrates for MsrBs. The chemical structures of some molecules described above are reported in Fig. 4.

5. Conclusions and Perspectives The ubiquitous distribution of Msrs suggests that they play important roles in the homeostasis of organisms, by intervening, most likely, in the defence against oxidative stress, through two major mechanisms. On the one hand, they directly repair the oxidative damage inflicted to proteins at the level of L-Met residues. On the other, they participate in the reductive arm of a cyclic mechanism where protein-bound LMet residues that have been oxidized to the sulfoxide by toxic oxidizing species are reduced back to L-Met, with the net result of scavenging and detoxifying potentially harmful reactive species. In other words, exposed L-Met residues in proteins may act as scavengers of oxidants and Msrs bring back the oxidized L-Met residues to the reduced state for another round of scavenging. The selective involvement of non-critical, surfaceexposed L-Met residues in proteins into this cycle would prevent oxidants from reaching other sensible components of the same or of other proteins and/or cellular components [126]. This hypothesis was corroborated by the evidence showing that the msrA null mutants of Saccharomyces cerevisiae [127] and mouse [76] are more susceptible to accumulation of oxidized L-Met residues than wild-type strains and, conversely, that overexpression of MsrA induces in yeast a drastic decrease in the levels of cellular L-Met-(O) [127]. The importance of MsrA in conferring to the cell resistance to oxidative stress has also been documented in studies showing that overexpression of this enzyme in the T-

Discovery of Methionine Sulfoxide Reductase Enzymes

lymphocytes line MOLT-4 [127], in Drosophila melanogaster [128], in the neuronal line PC12 [129] and in the human fibroblasts line WI-38 SV40 [130] leads to an increased survival after exposure to toxic levels of several oxidants. Again, msrA/ msrB double null mutant Saccharomyces cerevisiae [131] and msrB null mutant E. coli [56] exhibit an increased sensitivity to oxidation induced by exogenous oxidants. Furthermore, the increase in intracellular content of oxidized proteins that occurs during aging [132] has been correlated with the onset of typical pathologies of advanced age, such as the degeneration of macula lutea and cataract [133]. It is supposed that the increased sensitivity to oxidative stress during aging and in diseases typical of elderly is also a consequence of decreased levels of expression and the loss of Msr activity, as studies performed on rats seem to indicate [134,135]. The limited ability to reduce L-Met-(O) would decrease the efficiency of the cell defence systems that have evolved to counteract oxidative stress, with the consequent onset of degenerative pathologies such as systemic lupus erythematosus [136], Alzheimers disease [137], Parkinsons disease [138], emphysema [22,139] and cataract [140]. The expression of the selenoenzyme MsrB1 is strongly influenced by the intake of dietary selenium and aging, as observed in mice alimented with a selenium-deficient diet [141,142], in which high levels of oxidized proteins have also been detected [141]. These observations could be related to the imbalance of selenium status that occur during aging in humans, and that may lead to increased susceptibility to various age-related degenerative diseases, characterised by the impairment of the brain function such as Alzheimers disease, and in which high levels of oxidative damage were observed [143]. Surprisingly, it has been recently demonstrated that MsrA can exert an oxidase activity towards L-Met, that is converted to L-Met-S-(O) [144–146]. The proposed mechanism of this reaction, that takes place at high concentrations of L-Met and can also occur in the presence of the natural reducing system, involves the sulfenic acid generated by the oxidation of the catalytic Cys, and the oxidase function is limited to those MsrAs that are equipped with two recycling cysteines, where the intermediate disulfide bond involving catalytic and recycling cysteines produced in the second reducing step does not accumulate, and the sulfenic acid can be available for the reaction (for a detailed explanation, see Fig. 2). Furthermore it has been shown that MsrA is capable of catalysing the oxidation (and the reduction) of some L-Met residues in a-synuclein [144], a1-antitrypsin and calmodulin [146]. The biological role of this additional catalytic property of MsrA has yet to be investigated, but the double ability in oxidizing and reducing could represent a novel regulatory mechanism for protein function. In fact, many important biological processes could be regulated by the cyclic interconversion of L-Met and L-Met-(O) inserted in enzymes and other proteins with non-catalytic function [135,147]. Thus, Msr enzymes could play a role in cellular phenomena such as the adhesion of bacteria to cell mem-

branes [53], the action of hormones and plasma proteins [18,148], the transduction of signals that mediate opening and closing of potassium channels [149,150] and the operation of the calmodulin-dependent Ca21-ATPase [151]. It should be noted, however, that not always the oxidation of L-Met residues inhibits the biological properties of the proteins; in some cases the oxidation of certain L-Met residues is critical for the activation of the protein, such as for the calmodulin-dependent protein kinase II [152], and the transcriptional factor HypT [153]. Within this scenario, it is therefore clear that an imbalance of the ratio L-Met/L-Met-(O) could impact on cell metabolism, contributing to the onset of diseases related to the alteration of the affected cellular processes. On the basis of the above mentioned evidence, the knowledge that has been gained in the past few decades through the detailed characterisation of Msr enzymes represents an essential starting point for further research aimed at identifying the biochemical bases of certain serious, and still incurable, diseases, and at developing effective strategies to defeat them. In addition to sulindac and the other drugs described above, several other natural and synthetic compounds with pharmacological activity contain a methyl-sulfoxide group, such as the vasodilator flosequinan, whose reduction has been shown to take place in rat extract [154], and the inhibitors of platelet aggregation 1-[dithio alk(en)yl]-propyl-methyl sulfoxides‡‡ from onion [155,156], which could be substrates for Msrs. Moreover, some drugs and poisons that contain a methyl-sulfide group are metabolised in the organism through the oxidation to the sulfoxide, such as the inotropic agent enoximone [157], the lincosamide antibiotic clindamycin [158], the neuroleptic thioridazine (that is transformed into mesoridazine) [159], the dopaminergic alkaloid pergolide [160], the anthelmintic triclabendazole [161], and the organophosphorus pesticide fenthion [162]. Thanks to their potential ability of reducing the sulfoxide group of these compounds, Msrs could be involved in their pharmacodynamics and pharmacokinetics. The imbalance in the expression or in the activity of Msrs, typical of the elderly and of some diseases, could also induce alterations in the activity and metabolic pathways of these drugs. The existence of an enzymatic system that catalyse the reduction of molecules that contain one or more methylsulfoxide groups in their structure should be taken into account during the design and development of new pharmaceutical compounds. In recent years, certain types of enantiomerically pure chiral sulfoxides have found many applications in organic synthesis, thanks to their potential ability to act as chiral auxiliaries, and to drive the enantioselective formation of C–C bonds (nucleophilic addition and cycloaddition), but also in the reduction of carbonyl compounds and olefins. The industrial

‡‡

chemical formula: CH3–CH2–CH–(S–S–R)–SO–CH3 (where R could be: –CH3, –CH2–CH2–CH3, –CH–(OCH3)–CH2–CH3, cis/trans –CH=CH–CH3).

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preparation of chiral sulfoxides in optically pure form is performed with methods based on asymmetric oxidation, such as those catalysed by metals or that involve chiral auxiliaries themselves [163,164]. An alternative is the kinetic resolution of the racemic mixture by an enantiospecific reaction which involves the transformation of only one of the two enantiomers [165]. These methods, which yield products with very high optical purity, involve the use of hazardous reagents and high amounts of organic solvents. On the contrary, kinetic resolution carried out by an enzymatic system would not pose any major problem of environmental impact: in fact, enzyme-based systems are being increasingly employed for the synthesis of chiral molecules, with the perspective of promoting the development of a sustainable chemistry (the so-called green chemistry) [166,167]. For these reasons, Msrs could also find application as novel tools for the preparation of sulfoxides in enantiomeric pure form.

Acknowledgements This work was supported with FAR funds from the University of Pavia to GM, and with funds of the Fondazione Cariplo, Italy, grant no. 2011-2099. Authors would like to thank Prof. Herbert Weissbach for his help in revising the manuscript. Authors declare that they have no conflict of interest.

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