PDBsum entry: 2quu

Lyase

PDB id: 2quu

Contents

Protein chains

349 a.a. *

Ligands

13P ×4

Waters ×2230

* Residue conservation analysis

PDB id:

2quu

Name:

Lyase

Title:

Dihydroxyacetone phosphate schiff base intermediate in mutant fructose-1,6-bisphosphate aldolase from rabbit muscle

Structure:

Fructose-bisphosphate aldolase a. Chain: a, b, c, d. Synonym: muscle-type aldolase. Engineered: yes. Mutation: yes

Source:

Oryctolagus cuniculus. Rabbit. Organism_taxid: 9986. Gene: aldoa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.98Å R-factor: 0.157 R-free: 0.195

Authors:

M.St-Jean,J.Sygusch

Key ref:

M.St-Jean and J.Sygusch (2007). Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase. J Biol Chem, 282, 31028-31037. PubMed id: 17728250 DOI: 10.1074/jbc.M704968200

Date:

06-Aug-07 Release date: 28-Aug-07

Protein chains

P00883  (ALDOA_RABIT) -  Fructose-bisphosphate aldolase A

Seq: 364 a.a.

Struc: 349 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.4.1.2.13 - Fructose-bisphosphate aldolase.
• Reaction: D-fructose 1,6-bisphosphate = glycerone phosphate + D-glyceraldehyde 3-phosphate

D-fructose 1,6-bisphosphate = glycerone phosphate (Bound ligand (Het Group name = 13P) matches with 90.00% similarity) + D-glyceraldehyde 3-phosphate

• Cofactor: Zinc

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

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (2 terms)
• Biochemical function: catalytic activity (3 terms)

reference

DOI no: 10.1074/jbc.M704968200

J Biol Chem 282:31028-31037 (2007)

PubMed id: 17728250

Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase.

M.St-Jean, J.Sygusch.

ABSTRACT

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys-146 to Met aldolase were solved in complex with dihydroxyacetone-phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys-229 in native aldolase. Mutation of conserved active site residue Lys-146 to Met drastically decreases activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues, 360-363. Attachment positions the conserved C-terminal Tyr-363 hydroxyl within 2.9 A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr-363 is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by Lys-146 positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in presence of phosphate in the native structure corroborates Tyr-363 interaction with Lys-146 and is consistent with transient C-terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.

Selected figure(s)

Figure 4.
FIGURE 4. C terminus binding upon iminium formation in the active site of a subunit of the K146M mutant aldolase. The bound C terminus is shown superposed onto an alternate conformation of water molecules (cyan spheres) and Lys^107 side chain (cyan). Occupancy of C-terminal residues 360-363 was refined to 0.5 with water molecules accounting for the remaining electron density shown. Notably, the Tyr^363 hydroxyl is positioned at 2.9 Å from the DHAP C3 and in an orientation that would favor the stereospecific pro(S) -proton abstraction of the iminium (black dashes). Tyr^363 carboxylate binds Lys^107 whose side chain conformation is identical to that observed in native structures. Difference electron density was calculated from a 1.98-Å simulated annealing F[o] - F[c] omit map encompassing C-terminal residues 358-363 and contoured at 2.5 .

Figure 5.
FIGURE 5. Interactions made by the C-terminal region in the active site of muscle aldolase. A, C-terminal residues 361-363 interact with Ser^38, Arg^42, Lys^107, and Arg^148 in the active site of the K146M-DHAP structure. B, superposition of WT-DHAP (cyan), K146M-DHAP (yellow), and WT-P[i] (pink) structures. In the WT-DHAP structure, Ser^38 interacts with Lys^107 via two bridging water molecules, W3 and W4. Active site entry by the C terminus in K146M-DHAP and WT-P[i] structures displaces W3 and W4. Outwards movement of the flanking helix (residues 33-45) along its helical axis precludes a close contact that would result between Ser^38 side chain and Tyr^363 C upon active site binding.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 31028-31037) copyright 2007.

Figures were selected by an automated process.