4b5v

Publication Abstract from PubMed

Crystal structures of divalent metal dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8 - 2.0 A resolution. The enzyme-substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates water molecule, W2, bound to the divalent metal ion initiates C3-C4 bond cleavage. Binding mode of the aldehyde donor delineated a solvent filled capacious binding locus lined with predominantly hydrophobic residues. 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. Arg70 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 Arg70 stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, the pyruvate enolate upon aldol cleavage, as well as supporting 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 dissociation constant towards the pyruvate enolate analogue, oxalate, in the Arg70Ala 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 decarboyxlation of oxaloacetate.

Crystal structure of reaction intermediates in pyruvate Class II aldolase: Substrate Cleavage, Enolate Stabilization and Substrate Specificity.,Coincon M, Wang W, Sygusch J, Seah SY J Biol Chem. 2012 Aug 20. PMID:22908224^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.