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CSA entry for 2esd
Original Entry
Title:
Oxidoreductase
Compound:
Crystal structure of thioacylenzyme intermediate of an nadp
dependent aldehyde dehydrogenase
Mutant:
Yes
UniProt/Swiss-Prot:
Q59931-GAPN_STRMU
EC Class:
1.2.1.9
Other CSA Entries:
Overview of all sites for 2esd
Homologues of 2esd
Entries for UniProt/Swiss-Prot: Q59931
Entries for EC: 1.2.1.9
Other Databases:
PDB entry: 2esd
PDBsum entry: 2esd
UniProt/Swiss-Prot: Q59931
IntEnz entry: 1.2.1.9
Literature Report:
Introduction:
NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans (GAPN) is a non-phosphorylating, CoA-independent member of the aldehyde dehydrogenase (ALDH) family. It catalyses the irreversible oxidation of glyceraldehyde-3-phosphate (G3P) into 3-phosphoglycerate (3-PGA) in the presence of NADP via a two step mechanism.
Mechanism:
GAPN uses an ordered sequential two-step mechanism for catalysis - acylation followed by deacylation.

First, NADP+ binds to the enzyme, inducing a conformational change in the active site, resulting in the activation of a competent enzyme. The positive charge of the nicotinamide ring of NADP+, the amide peptide nitrogens of residues Cys284 and Thr285, and possibly the involvement of other structural elements stabilise the thiolate form of Cys284.

G3P binding leads to formation of a thiohemiacetal intermediate via a nucleophilic attack by the Cys284 thiolate. This is followed by an oxidoreduction process in which a hydride is transferred from the intermediate to the nicotinamide ring of NADP+ forming a thioacylenzyme intermediate. This is able to take place without catalytic assistance due to the presence of an oxianion hole, formed by Asn154 and the amide nitrogen of Cys284, interacting with the C1 oxygen of the substrate, keeping the oxygen deprotonated. The oxyanion hole is also involved in the stabilisation of the tetrahedral thioacyl intermediate.

Then deacylation occurs, where Glu250 orientates and activates water which nucleophilically attacks the C1 carbon of the thioacyl intermediate, leading to an acid product and, finally, the release of the reduced cofactor (NADPH). The isomerisation and movement of the nicotinamide ring of NADPH after the oxidoreduction stage is required to allow Glu250 to perform its role in deacylation.
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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
1476 0
Substrate
ElectrostaticResidue
On NADP+ binding, a local conformational rearrangement of the nicotinamide subsite, the reorientation of the lateral side chains of Cys284 and Glu250, and the repositioning of the nicotinamide ring of NADP+ is induced. This makes decreases the pkA of Cys284 and makes the residue more accessible to the substrate. The positive charge of the cofactor is involved in stabilising the thiolate form of the catalytic Cys. NADP+ undergoes hydride transfer from the hemiacetal intermediate to the C4 position of the cofactor during the oxidoreduction process in the acylation step, and dissociates from the enzyme last in its reduced form.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16503652 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASNA 154 154Sidechain
ElectrostaticTransition state
Asn154-ND2 group and the amide nitrogen of Cys284 interact with the C1 oxygen of the substrate to form an oxianion hole which is required of hydride transfer and involved in the stabilisation of the intermediates.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11306027 Current protein Mutagenesis of residue
PubMed ID 16503652 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ALAA 250 250Sidechain
ElectrostaticWater
Acid/baseWater
Glu250 orientates and activates water which acts as a nucleophile and attacks the C1 carbon of the thioacylenzyme intermediate, leading to the formation an acid product of 3-PGA and the release of the reduced cofactor. At basic pH, deacylation is catalysed by the chemical activation of the water molecule attacking the thioacyl intermediate via the abstraction of a proton by Glu250 acting as a base catalyst.
Evidence from paper Evidence concerns Evidence type
PubMed ID 10727225 Current protein Mutagenesis of residue
PubMed ID 10727225 Current protein Kinetic studies
PubMed ID 10727225 Current protein Ligand is essential for catalysis

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 284 284Sidechain, Backbone amide
NucleophileSubstrate
ElectrostaticTransition state
In thiolate form, Cys284 acts as a nucleophile and attacks C1 of G3P substrate to form a thiohemiacetal enzyme intermediate in the acylation process. The amide nitrogen of Cys284, in conjunction with Asn154, interacts with the C1 oxygen of the substrate to form an oxianion hole which is required for hydride transfer and involved in the stabilisation of the intermediates.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11306027 Current protein Ligand is essential for catalysis
PubMed ID 10864505 Current protein Mutagenesis of residue
PubMed ID 16503652 Current protein Residue is covalently bound to intermediate, based on structural data
Notes:

References:
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