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Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2′ phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs. Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.

Peptide methionine sulfoxide reductase mediates the reduction of protein sulfoxide methionyl residues back to methionines and could thus be implicated in the antioxidant defence of organisms. Hexagonal crystals of the Escherichia coli enzyme (MsrA) were obtained by the hanging-drop vapour-diffusion technique. They belong to space group P6(5)22, with unit-cell parameters a = b = 102.5, c = 292.3 A, gamma = 120 degrees. A native data set was collected at 1.9 A resolution. Crystals of selenomethionine-substituted MsrA were also grown under the same crystallization conditions. A three-wavelength MAD experiment has led to the elucidation of the positions of the Se atoms and should result in a full structure determination.

The NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (abbreviated Sm-ALDH) belongs to the aldehyde dehydrogenase (ALDH) family. Its catalytic mechanism proceeds via two steps, acylation and deacylation. Its high catalytic efficiency at neutral pH implies prerequisites relative to the chemical mechanism. First, the catalytic Cys284 should be accessible and in a thiolate form at physiological pH to attack efficiently the aldehydic group of the glyceraldehyde-3-phosphate (G3P). Second, the hydride transfer from the hemithioacetal intermediate toward the nicotinamide ring of NADP should be efficient. Third, the nucleophilic character of the water molecule involved in the deacylation should be strongly increased. Moreover, the different complexes formed during the catalytic process should be stabilised. The crystal structures presented here (an apoenzyme named Apo2 with two sulphate ions bound to the catalytic site, the C284S mutant holoenzyme and the ternary complex composed of the C284S holoenzyme and G3P) together with biochemical results and previously published apo and holo crystal structures (named Apo1 and Holo1, respectively) contribute to the understanding of the ALDH catalytic mechanism. Comparison of Apo1 and Holo1 crystal structures shows a Cys284 side-chain rotation of 110 degrees, upon cofactor binding, which is probably responsible for its pK(a) decrease. In the Apo2 structure, an oxygen atom of a sulphate anion interacts by hydrogen bonds with the NH2 group of a conserved asparagine residue (Asn154 in Sm-ALDH) and the Cys284 NH group. In the ternary complex, the oxygen atom of the aldehydic carbonyl group of the substrate interacts with the Ser284 NH group and the Asn154 NH2 group. A substrate isotope effect on acylation is observed for both the wild-type and the N154A and N154T mutants. The rate of the acylation step strongly decreases for the mutants and becomes limiting. All these results suggest the involvement of Asn154 in an oxyanion hole in order to stabilise the tetrahedral intermediate and likely the other intermediates of the reaction. In the ternary complex, the cofactor conformation is shifted in comparison with its conformation in the C284S holoenzyme structure, likely resulting from its peculiar binding mode to the Rossmann fold (i.e. non-perpendicular to the plane of the beta-sheet). This change is likely favoured by a characteristic loop of the Rossmann fold, longer in ALDHs than in other dehydrogenases, whose orientation could be constrained by a conserved proline residue. In the ternary and C284S holenzyme structures, as well as in the Apo2 structure, the Glu250 side-chain is situated less than 4 A from Cys284 or Ser284 instead of 7 A in the crystal structure of the wild-type holoenzyme. It is now positioned in a hydrophobic environment. This supports the pK(a) assignment of 7.6 to Glu250 as recently proposed from enzymatic studies.

Background: Peptide methionine sulphoxide reductases catalyze the reduction of oxidized methionine residues in proteins. They are implicated in the defense of organisms against oxidative stress and in the regulation of processes involving peptide methionine oxidation/reduction. These enzymes are found in numerous organisms, from bacteria to mammals and plants. Their primary structure shows no significant similarity to any other known protein. Results: The X-ray structure of the peptide methionine sulphoxide reductase from Escherichia coli was determined at 3 A resolution by the multiple wavelength anomalous dispersion method for the selenomethionine-substituted enzyme, and it was refined to 1.9 A resolution for the native enzyme. The 23 kDa protein is folded into an α/β roll and contains a large proportion of coils. Among the three cysteine residues involved in the catalytic mechanism, Cys-51 is positioned at the N terminus of an α helix, in a solvent-exposed area composed of highly conserved amino acids. The two others, Cys-198 and Cys-206, are located in the C-terminal coil. Conclusions: 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 α 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.