PDBsum entry 1qi1

Oxidoreductase

PDB id: 1qi1

Contents

Protein chains

474 a.a. *

Ligands

NAP ×4

G3H ×3

G3P

* Residue conservation analysis

PDB id:

1qi1

Name:

Oxidoreductase

Title:

Ternary complex of an NADP dependent aldehyde dehydrogenase

Structure:

Protein (NADP-dependent nonphosphorylating glyceraldehyde- 3-phosphate dehydrogenase). Chain: a, b, c, d. Engineered: yes. Mutation: yes. Other_details: NADP, d-glyceraldehyde-3-phosphate

Source:

Streptococcus mutans. Organism_taxid: 1309. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Tetramer (from PQS)

Resolution:

3.00Å R-factor: 0.242 R-free: 0.283

Authors:

D.Cobessi,F.Tete-Favier,S.Marchal,G.Branlant,A.Aubry

Key ref:

D.Cobessi et al. (2000). Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans. J Mol Biol, 300, 141-152. PubMed id: 10864505 DOI: 10.1006/jmbi.2000.3824

Date:

02-Jun-99 Release date: 10-Jan-01

Protein chains

Q59931  (GAPN_STRMU) -  NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans serotype c (strain ATCC 700610 / UA159)

Seq: 475 a.a.

Struc: 474 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.2.1.9 - glyceraldehyde-3-phosphate dehydrogenase (NADP(+)).
• Reaction: D-glyceraldehyde 3-phosphate + NADP⁺ + H2O = (2R)-3-phosphoglycerate + NADPH + 2 H⁺

D-glyceraldehyde 3-phosphate (Bound ligand (Het Group name = G3H) corresponds exactly) + NADP(+) (Bound ligand (Het Group name = NAP) corresponds exactly) + H2O = (2R)-3-phosphoglycerate + NADPH + 2 × H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1006/jmbi.2000.3824

J Mol Biol 300:141-152 (2000)

PubMed id: 10864505

Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans.

D.Cobessi, F.Tête-Favier, S.Marchal, G.Branlant, A.Aubry.

ABSTRACT

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.

Selected figure(s)

Figure 1.
Figure 1. Stereo view of the superposition of the Apo1 and Apo2 catalytic sites. The Asn154, Glu250, Cys284, SO[4]a and SO[4]b are displayed in ball-and-stick using MOLSCRIPT [Kraulis 1991] and Raster3D [Meritt and Murphy 1994]. Traces of the described residues forming the hydrophobic environment of Glu250 in Apo2 are displayed. The Glu250 side-chain in Apo1 (Apo2) is coloured green (red). Accessibility of Glu250 in Apo2 is 2 Å2.

Figure 4.
Figure 4. (a) Stereo view of NADP in a 3F[o] - 2F[c] electron density map contoured at 1.2s generated using TURBO-FRODO [Roussel and Cambillau 1991] for the C284S structure. (b) Stereo view of NADP in a 3F[o] - 2F[c] electron density map contoured at 1.2s generated using TURBO-FRODO [Roussel and Cambillau 1991] for the ternary complex.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 300, 141-152) copyright 2000.

Figures were selected by an automated process.