1qi6
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SECOND APO FORM OF AN NADP DEPENDENT ALDEHYDE DEHYDROGENASE WITH GLU250 SITUATED 3.7 A FROM CYS284
OverviewOverview
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.
About this StructureAbout this Structure
1QI6 is a Single protein structure of sequence from Streptococcus mutans with SO4 as ligand. Active as Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)), with EC number 1.2.1.9 Full crystallographic information is available from OCA.
ReferenceReference
Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans., Cobessi D, Tete-Favier F, Marchal S, Branlant G, Aubry A, J Mol Biol. 2000 Jun 30;300(1):141-52. PMID:10864505
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