PDBsum entry 1uxt

Oxidoreductase

PDB id: 1uxt

Contents

Protein chain

499 a.a. *

Ligands

G1P

NAD

Metals

_NA

Waters ×268

* Residue conservation analysis

PDB id:

1uxt

Name:

Oxidoreductase

Title:

Structural basis for allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (gapn) from thermoproteus tenax

Structure:

Glyceraldehyde-3-phosphate dehydrogenase (NADP+). Chain: a. Engineered: yes. Mutation: yes. Other_details: glucose 1-phosphate and NAD bound noncovalently

Source:

Thermoproteus tenax. Organism_taxid: 2271. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.20Å R-factor: 0.224 R-free: 0.242

Authors:

E.Lorentzen,R.Hensel,E.Pohl

Key ref:

E.Lorentzen et al. (2004). Structural Basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-Phosphate dehydrogenase from Thermoproteus tenax. J Mol Biol, 341, 815-828. PubMed id: 15288789 DOI: 10.1016/j.jmb.2004.05.032

Date:

01-Mar-04 Release date: 05-Aug-04

Protein chain

O57693  (GAPN_THETE) -  NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax

Seq: 501 a.a.

Struc: 499 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.2.1.90 - glyceraldehyde-3-phosphate dehydrogenase [NAD(P)(+)].
• Reaction:
  1. D-glyceraldehyde 3-phosphate + NADP⁺ + H2O = (2R)-3- phosphoglycerate + NADPH + 2 H⁺
  2. D-glyceraldehyde 3-phosphate + NAD⁺ + H2O = (2R)-3-phosphoglycerate + NADH + 2 H⁺

D-glyceraldehyde 3-phosphate (Bound ligand (Het Group name = G1P) matches with 62.50% similarity) + NADP(+) + H2O (Bound ligand (Het Group name = NAD) matches with 91.67% similarity) = (2R)-3- phosphoglycerate + NADPH + 2 × H(+)

D-glyceraldehyde 3-phosphate (Bound ligand (Het Group name = G1P) matches with 62.50% similarity) + NAD(+) + H2O (Bound ligand (Het Group name = NAD) corresponds exactly) = (2R)-3-phosphoglycerate + NADH + 2 × H(+)

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

reference

DOI no: 10.1016/j.jmb.2004.05.032

J Mol Biol 341:815-828 (2004)

PubMed id: 15288789

Structural Basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-Phosphate dehydrogenase from Thermoproteus tenax.

E.Lorentzen, R.Hensel, T.Knura, H.Ahmed, E.Pohl.

ABSTRACT

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the modified glycolytic pathway of this organism. In contrast to other members of the ALDH superfamily, GAPN from T.tenax (Tt-GAPN) is regulated by a number of intermediates and metabolites. In the NAD-dependent oxidation of GAP, glucose 1-phosphate, fructose 6-phosphate, AMP and ADP increase the affinity for the cosubstrate, whereas ATP, NADP, NADPH and NADH decrease it leaving, however, the catalytic rate virtually unaltered. As we show here, the enzyme also uses NADP as a cosubstrate, displaying, however, unusual discontinuous saturation kinetics indicating different cosubstrate affinities and/or reactivities of the four active sites of the protein tetramer caused by cooperative effects. Furthermore, in the NADP-dependent reaction the presence of activators decreases the overall S0.5 and increases Vmax by a factor of 3. To explore the structural basis for the different effects of both pyridine nucleotides we solved the crystal structure of Tt-GAPN in complex with NAD at 2.2 A resolution and compared it to the binary Tt-GAPN-NADPH structure. Although both pyridine nucleotides show a similar binding mode, NADPH appears to be more tightly bound to the protein via the 2' phosphate moiety. Moreover, we present four co-crystal structures with the activating molecules glucose 1-phosphate, fructose 6-phosphate, AMP and ADP determined at resolutions ranging from 2.3 A to 2.6 A. These crystal structures reveal a common regulatory site able to accommodate the different activators. A phosphate-binding pocket serves as an anchor point ensuring similar binding geometry. The observed conformational changes upon activator binding are discussed in terms of allosteric regulation. Furthermore, we present a crystal structure of Tt-GAPN in complex with the substrate D-GAP at 2.3 A resolution, which allows us to analyse the structural basis for substrate binding, the mechanism of catalysis as well as the stereoselectivity of the enzymatic reaction.

Selected figure(s)

Figure 2.
Figure 2. (a) NADP saturation of Tt-GAPN in the absence of activator. The insert shows the concentration range of 0-0.5 mM NADP. (b) NADP saturation of Tt-GAPN in the absence (sB) and presence of 50 µM G1P (cD-).

Figure 5.
Figure 5. Hydrogen-bonding network involving the C-terminal carboxyl of a symmetry equivalent monomer, the phosphate moiety of an activator, the carbonyl of Arg72 and a well ordered water molecule. The binding of activators leads to a considerable fixation of the otherwise partially disordered C terminus of the protein.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 341, 815-828) copyright 2004.

Figures were selected by an automated process.