Characterization of the Amino Acids Involved in Substrate Specificity of Methionine Sulfoxide Reductase A

Abstract

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i. Methionine is one of the amino acids in proteins that is the most sensitive to reactive oxygen species (1). It is converted into methionine sulfoxide (MetSO), 2 the function of which is a mixture of two epimers at the sulfur atom, i.e. Met-S-SO and Met-R-SO (2). Formation of MetSO may impair the biological function of the oxidized proteins, depending on the location of the MetSO in the protein. There exist two structurally unrelated classes of methionine sulfoxide reductases (Msrs) in most organisms, called MsrA and MsrB, which selectively reduce free or protein-bound Met-S-SO and Met-R-SO, respectively. Msrs are described to exert various biological functions in vivo (3-5). They can (i) repair oxidized proteins, and thus, may regulate their function, (ii) play an antioxidant role as oxidation of surface methionine residues is considered as a mechanism that scavenges reactive oxygen species without modification of the properties of proteins, and (iii) play a role in the virulence of some bacteria.The catalytic mechanism of both classes of Msrs characterized to date (6 -9) is composed of three steps including: 1) a reductase step consisting of a nucleophilic attack of the catalytic Cys residue on the sulfur atom of the sulfoxide substrate that leads to formation of a sulfenic acid intermediate and release of 1 mol of Met/mol of enzyme, 2) formation of an intradisulfide bond between the catalytic Cys and a recycling Cys with a concomitant release of 1 mol of water, and 3) reduction of the Msr disulfide bond by thioredoxin (Trx) that leads to regeneration of the reduced form of Msr and to formation of oxidized Trx. The catalytic mechanism is in agreement with the kinetic mechanism, which was shown to be of ping-pong type for both classes of Msrs (9, 10). Moreover, for both classes of Msrs, the overall rate-limiting step is associated with the Trxrecycling process, whereas the rate of formation of the intradisulfide bond is governed by that of the reductase step, the rate of which is fast (11,12).A theoretical study of the reduction mechanism of sulfoxides by thiols has been recently investigated by quantum chemistry calculations, which supp...

Methionine Sulfoxide Reductase B Displays a High Level of Flexibility

“…Second, the structure of the N. meningitidis MsrA active site, described as a "mirror" of the MsrB active site, revealed that the presence of the sulfenic acid in the catalytic Cys51 does not induce any conformational change within the active site, in particular at Trp53, whose role is equivalent to that of Trp65. 13,15,18 Recently, the NMR structure of Bacillus subtilis MsrB, which possesses the catalytic Cys117 and the recycling Cys63, was deposited in the PDB by Zhang et al (PDB ID 1XM0; unpublished results) from the "Northeast Structural Genomics" project (Target SR10). The 10 models available in the PDB file show that the loops are highly flexible (Fig.…”

A Structural Analysis of the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

“…Altogether, these results are consistent with the recent enzymology studies done by our group. 22 In that context, the structural/molecular factors that contribute to the better binding of protein-bound MetSO rather than free MetSO remain to be characterized.…”

“…Recently, amino acids involved in the substrate specificity and in the catalysis of the reductase step of the N. meningitidis MsrA domain have been characterized by molecular enzymology approaches. 15,22 A scenario of the catalysis of the reductase step has been proposed. 15 In all the X-ray structures determined so far, the distance between Cys51 and Cys198 is too large to allow the formation of an intramolecular disulfide bridge.…”

Molecular Mechanisms of the Methionine Sulfoxide Reductase System from Neisseria meningitidis

“…Nonetheless, it is clear that the presence of the sulfoxide function is a prerequisite for binding to both MSRs. The active sites of MSRA and MSRB are both located on the surface of the enzymes and contain two mirror-image sub-sites which confer stereospecificity for the R or S stereoisomer of L-Met-O: (1) a hydrophilic pocket involving the stabilization of the protonated sulfoxide function of the substrate via hydrogen bonding; and (2) a hydrophobic pocket consisting essentially of the side chain of a Trp residue, which stabilizes the ε-methyl group of the substrate via its indole ring ( Figure 4 ) [ 9 , 13 , 28 , 29 ]. The selectivity of the enzymes for protein-bound Met-O versus free Met-O probably derives from a destabilizing effect, due to repulsive interactions with the charged amino and/or carboxyl groups of the free amino acid.…”