A Sulfenic Acid Enzyme Intermediate Is Involved in the Catalytic Mechanism of Peptide Methionine Sulfoxide Reductase fromEscherichia coli

Abstract

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. Aerobic metabolism produces a great number of activated oxygen species. These species can react with various targets including proteins. In particular, methionine residues can be oxidized into methionine sulfoxide (MetSO).1 Such modifications can alter the biological properties of the targeted proteins (1). For instance, this likely is the case for the ␣-proteinase inhibitor whose oxidation of a methionine residue decreases its affinity relative to its protease target (2) and also for calmodulin whose methionine oxidation leads to a decrease in the efficiency of activation of plasma membrane (3). On the other hand the fact that methionine modifications can be also restricted to only surface-exposed residues was interpreted as a way to protect cells against the action of reactive oxygen species (4). In vivo a ubiquitous enzyme named peptide methionine sulfoxide reductase (MsrA) exists, which reduces both free and protein bound MetSO (5, 6). The fact that the null mutants of both Escherichia coli and yeast showed increased sensitivity against oxidative damage and that overexpression of MsrA gave higher resistance to hydrogen treatment supports an essential role of MsrA in cell viability (7,8). Thus the important biological role attributed to MsrA in vivo justifies a study of the chemical mechanism of the reduction of MetSO by MsrA. The fact that MsrA activity necessitates a thioredoxin recycling system (9 -11) suggested a cysteine residue in the chemical catalysis. Recently, two groups have shown that mutating the invariant cysteine located in the conserved signature Gly-CysPhe-Trp resulted in total loss of enzyme activity (12, 13). Moreover Lowther et al. (13) presented convincing evidence of involvement of intra-thiol-disulfide exchanges in the catalytic mechanism. Based on their data they formulated a reaction mechanism requiring formation of a covalent tetracoordinate intermediate via a nucleophilic attack by the thiolate of the essential cysteine followed by breakdown of the intermediate by means of two thiol-disulfide exchanges, which leads to release of a methionine and a water molecule. In this mechanism, methionine release only occurs if the disulfide exchange is operative. In the present study we show that in fact the nucleophilic attack of the essential cysteine on MetSO leads to formation of a sulfenic acid enzyme intermediate with a concomitant release of methionine. Return of the active site to a reduced state is achieved in vivo via intra-disulfide exchange reactions involving two other cysteines and then by a thioredoxindependent recycling process. EXPERIMENTAL PROCEDURESSite-directed Mutagenesis, Production, and Purification of Wild Type and Mutant E. coli MsrAs-The E. coli strain ...

Kinetic Characterization of the Chemical Steps Involved in the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

“…The second one, which is represented by E. coli and B. taurus MsrAs, possesses three essential cysteines at positions 51, 198, and 206. In this case, only the Cys-198/Cys-206 disulfide bond was shown to be reduced by Trx (10). In fact, the reduction of the Cys-51/Cys-198 bond probably does not occur because of its nonaccumulation.…”

“…Met quantification in the resulting supernatant was carried out by reverse-phase chromatography as described previously in Ref. 10, with slight modifications: 100 l were injected onto a 4.6 ϫ 250 mm Atlantis dC18 reverse-phase column (Waters) on an AKTA explorer system (Amersham Biosciences), equilibrated with H 2 O, 7.5% acetonitrile, 0.1% trifluoroacetic acid. Met was eluted isocratically after MetSO.…”

“…Quantification of the Free Cysteine Content with 5,5Ј-Dithiobis(2-nitro)benzoate (DTNB)-Cysteine content of MsrA and Trx was determined using DTNB under nondenaturing conditions in buffer A (50 mM Tris-HCl, 2 mM EDTA, pH 8) as described previously (10).…”

Free methionine-( R )-sulfoxide reductase from Escherichia coli reveals a new GAF domain function

“…The collapse of the resulting intermediate would result in the formation of a Cys sulfenic acid (Cys-94-SOH). Our attempts to verify the formation of this intermediate by dimedone treatment were undoubtedly thwarted by the inability of the dimedone molecule (140 Da) to access the enclosed cavity during catalysis (29). The lack of conservation of Cys-84 in some organisms (SI Fig.…”

SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation

“…This enzyme was shown to bind its substrate methionine sulfoxide via the thiol group of a highly conserved cysteine residue. After the release of methionine the active-site cysteine has to be re-reduced by a thioredoxin-regenerating system or DTT (Boschi-Muller et al, 2000;Lowther et al, 2000).…”