ABSTRACT
2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apo-structure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.
Subject(s)
AAA Domain , Aldehyde-Lyases/chemistry , Protein Conformation , Zymomonas/enzymology , Aldehyde-Lyases/genetics , Aldehyde-Lyases/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Databases, Protein , Glyceric Acids/metabolism , Models, Molecular , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
Zymomonas mobilis ZM4 is an organism optimized for ethanol production which uses the Entner-Doudoroff (ED) pathway for the breakdown of glucose. The key enzyme in this process is 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which produces glyceraldehyde 3-phosphate and pyruvate. In order to provide a molecular background for the KDPG aldolase from this ethanologenic organism (zmKDPG aldolase), the ZMO0997 gene of Z. mobilis ZM4 coding for zmKDPG aldolase was cloned and expressed and the purified protein was crystallized from 25%(w/v) polyethylene glycol 3350 and 0.1 M bis-tris pH 5.5. Diffraction data were collected to 1.8 A resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 83.0, c = 117.2 A. A trimeric zmKDPG aldolase molecule was present in the asymmetric unit, resulting in a crystal volume per unit protein weight of 2.40 A(3) Da(-1) and a solvent content of 48%.