Bacillus subtilis possesses two similar putative phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding genes, gap (renamed gapA) and gapB. A gapA mutant was unable to grow on glycolytic carbon sources, although it developed as well as the wild-type strain on gluconeogenic carbon sources. A gapB mutant showed the opposite phenotype. Purified GapB showed a 50-fold higher GAPDHase activity with NADP ؉ than with NAD ؉ , with K m values of 0.86 and 5.7 mM, respectively. lacZ reporter gene fusions revealed that the gapB gene is transcribed during gluconeogenesis and repressed during glycolysis. Conversely, gapA transcription is 5-fold higher under glycolytic conditions than during gluconeogenesis. GAPDH activity assays in crude extracts of wild-type and mutant strains confirmed this differential expression pattern at the enzymatic level. Genetic analyses demonstrated that gapA transcription is repressed by the yvbQ (renamed cggR) gene product and indirectly stimulated by CcpA. Thus, the same enzymatic step is catalyzed in B. subtilis by two enzymes specialized, through the regulation of their synthesis and their enzymatic characteristics, either in catabolism (GapA) or in anabolism (GapB). Such a dual enzymatic system for this step of the central carbon metabolism is described for the first time in a nonphotosynthetic eubacterium, but genomic analyses suggest that it could be a widespread feature.Glycolysis is the main pathway for degradation of carbohydrates and is found in nearly all groups of organisms. The formation of the final product of glycolysis, pyruvate, from glucose is achieved by nine enzymatic steps, most of which function in the reverse direction during gluconeogenesis. The phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1 occupies a pivotal role in the Embden-Meyerhoff pathway not only in glycolysis but also in gluconeogenesis because of the reversibility of the oxidation of glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (1,3dPG). In plants, two distinct types of phosphorylating GAPDH co-exist: (i) a strictly NAD-dependent cytoplasmic GAPDH involved in glycolysis and gluconeogenesis; and (ii) a chloroplastic GAPDH, which is involved in photosynthetic CO 2 assimilation and exhibits a dual coenzyme specificity with a preference for NADP (1, 2).Recently, two gap genes, named gap1 and gap2, have been characterized in the cyanobacterium Synechocystis sp. PPC 6803. The NAD-dependent enzyme Gap1 was reported to be essential for glycolytic glucose breakdown, whereas the enzyme Gap2, which exhibits dual coenzyme specificity, was shown to be operative in the photosynthetic Calvin cycle and in nonphotosynthetic gluconeogenesis (3). Thus, at least in some photoautotrophic bacterial species (in which the photosynthetic Calvin cycle and glycolysis/gluconeogenesis function in the same cellular compartment, in contrast to what happens in land plants and algae) two distinct GAPDHs, a strictly NAD-dependent one and the photosynthetic one, catalyze the two ...

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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 ...

PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/ MsrB) of N. meningitidis were produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding proteinbound MetSO more efficiently than free MetSO; 3) the carbon C␣ is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols.Peptide methionine sulfoxide reductase (MsrA) 1 activity is described as being involved in the virulence of the pathogens Escherichia coli, Streptococcus pneumoniae, Erwinia chrysanthemi, Mycoplasma genitalium, and Neisseria gonorrhoeae (1-4). Inspection of the alignment of the corresponding protein sequences shows that all possess in common a sequence that displays an MsrA activity. This MsrA activity has now been well characterized at the structural level (5, 6) and the enzymatic level (7). In particular, a sulfenic acid intermediate has been shown to be formed on Cys-51 of E. coli MsrA during the reduction of the sulfoxide function of methionine sulfoxide (MetSO). The active site can be represented as an open basin in which Cys-51, located at the N terminus of an ␣-helix, is accessible. Compared with the E. coli MsrA, the MsrAs from S. pneumoniae and from N. meningitidis or N. gonorrhoeae (called PILB) contain, in addition, an extension at the C terminus and at the C and N termini, respectively. This raised the question of the role of these extensions, in particular of the C-terminal extension. Sequence comparisons of the C-extension of PILB show amino acid identities with open reading frames of which no function has been...

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Sequence alignments show that the overall fold of the peptide methionine sulphoxide reductase from E. coli is likely to be conserved in many species. The characteristics observed in the Cys-51 environment are in agreement with the expected accessibility of the active site of an enzyme that reduces methionine sulphoxides in various proteins. Cys-51 could be activated by the influence of an alpha helix dipole. The involvement of the two other cysteine residues in the catalytic mechanism requires a movement of the C-terminal coil. Several conserved amino acids and water molecules are discussed as potential participants in the reaction.

Oxidation of methionine into methionine sulfoxide is associated with many pathologies and is described to exert regulatory effects on protein functions. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, have been described to reduce the S and the R isomers of the sulfoxide of methionine sulfoxide back to methionine, respectively. Although MsrAs and MsrBs display quite different x-ray structures, they share a similar, new catalytic mechanism that proceeds via the sulfenic acid chemistry and that includes at least three chemical steps with 1) the formation of a sulfenic acid intermediate and the concomitant release of methionine; 2) the formation of an intra-disulfide bond; and 3) the reduction of the disulfide bond by thioredoxin. In the present study, it is shown that for the Neisseria meningitidis MsrA, 1) the rate-limiting step is associated with the reduction of the Cys-51/Cys-198 disulfide MsrA bond by thioredoxin; 2) the formation of the sulfenic acid intermediate is very efficient, thus suggesting catalytic assistance via amino acids of the active site; 3) the rate-determining step in the formation of the Cys-51/ Cys-198 disulfide bond is that leading to the formation of the sulfenic intermediate on Cys-51; and 4) the apparent affinity constant for methionine sulfoxide in the methionine sulfoxide reductase step is 80-fold higher than the K m value determined under steady-state conditions.Methionine residues are easily oxidized to methionine sulfoxides (MetSO) 1 by reactive oxygen and nitrogen species produced either by aerobic metabolism or after exposure to various agents. This post-translation modification can provoke loss of protein function (1) and has been implicated in the aging process (for a review, see Ref.2). Therefore, organisms have developed various defense strategies. One consists of restoring the biological function of the modified MetSO proteins. This is the role of methionine sulfoxide reductases (Msr), which reduce MetSO back to methionine (Met). There exist two classes of Msrs, called MsrA and MsrB. The physiological role of MsrAs has been particularly well illustrated. For instance, mice lacking MsrA show an atypical walking pattern and have a reduced life span (3), whereas in contrast, overexpression of the msrA gene in the nervous system markedly extends the life span of the fruit fly Drosophila (4).MsrAs and MsrBs have been extensively characterized at both structural (5-9) and enzymatic levels (10 -14). Although MsrAs from Escherichia coli, Bos taurus, and Mycobacterium tuberculosis and MsrB from Neisseria gonohorreae show quite different three-dimensional x-ray structures and are specific for the S and the R isomers of the sulfoxide of MetSO, respectively, both structural classes of Msrs present a similar, new catalytic mechanism that includes a minimum of three chemical steps (Scheme 1). This is the case for MsrA and MsrB from Neisseria meningitidis (13). In the first step, a Michaelis complex is formed between Msr and MetSO that precedes the nucleophilic attac...

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.