4B5X

Crystal structures of divalent metal dependent pyruvate aldolase (HpaI), mutant D42A

Summary for 4B5X

• Entry DOI: 10.2210/pdb4b5x/pdb
• Related: 2V5J 2V5K 4B5S 4B5T 4B5U 4B5V 4B5W
• Descriptor: 4-HYDROXY-2-OXO-HEPTANE-1,7-DIOATE ALDOLASE, PHOSPHATE ION, GLYCEROL, ... (4 entities in total)
• Functional Keywords: lyase, catalytic mechanism
• Biological source: ESCHERICHIA COLI ATCC 8739
• Total number of polymer chains: 2
• Total formula weight: 57050.03

Authors

Coincon, M.,Wang, W.,Seah, S.Y.K.,Sygusch, J. (deposition date: 2012-08-07, release date: 2012-08-29, Last modification date: 2023-12-20)

Primary citation

Coincon, M.,Wang, W.,Sygusch, J.,Seah, S.Y.K.
Crystal Structure of Reaction Intermediates in Pyruvate Class II Aldolase: Substrate Cleavage, Enolate Stabilization and Substrate Specificity
J.Biol.Chem., 287:36208-, 2012

PubMed Abstract: Crystal structures of divalent metal-dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8-2.0 Å resolution. The enzyme·substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates that water molecule W2 bound to the divalent metal ion initiates C3-C4 bond cleavage. The binding mode of the aldehyde donor delineated a solvent-filled capacious binding locus lined with predominantly hydrophobic residues. The absence of direct interactions with the aldehyde aliphatic carbons accounts for the broad specificity and lack of stereospecific control by the enzyme. Enzymatic complex structures formed with keto acceptors, pyruvate, and 2-ketobutyrate revealed bidentate interaction with the divalent metal ion by C1-carboxyl and C2-carbonyl oxygens and water molecule W4 that is within close contact of the C3 carbon. Arg(70) assumes a multivalent role through its guanidinium moiety interacting with all active site enzymatic species: C2 oxygen in substrate, pyruvate, and ketobutyrate; substrate C4 hydroxyl; aldehyde C1 oxygen; and W4. The multiple interactions made by Arg(70) stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, and the pyruvate enolate upon aldol cleavage as well as support proton exchange at C3. This role is corroborated by loss of aldol cleavage ability and pyruvate C3 proton exchange activity and by a 730-fold increase in the dissociation constant toward the pyruvate enolate analog oxalate in the R70A mutant. Based on the crystal structures, a mechanism is proposed involving the two enzyme-bound water molecules, W2 and W4, in acid/base catalysis that facilitates reversible aldol cleavage. The same reaction mechanism promotes decarboxylation of oxaloacetate.

PubMed: 22908224
DOI: 10.1074/JBC.M112.400705

Experimental method

X-RAY DIFFRACTION (1.8 Å)