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.

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. The proposed chemical mechanism ofEscherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 → Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.

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. meningitidiswere 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 protein-bound MetSO more efficiently than free MetSO; 3) the carbon Cα 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.