Mechanism of the Class I KDPG aldolase

Abstract

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.

Figure 1

The sequence homology between E. coli KDPG and KDPGal aldolases using CLUSTALW alignment.

Figure 2

Monomer and trimer of KDPGal aldolase.

Figure 3

The E. coli KDPGal structure in cyan is essentially identical to the E. coli KDPG aldolase structure shown in purple.

Figure 4

(a) The co-complex of KDPGal aldolase with bound substrate; hydrogen bonds are shown as dotted lines. The residues discussed in the text are shown in stick and labeled. (b) The Fo-Fc electron density map contoured at 2.5Å. Phases were derived from a model prior to any addition of ligand or water to this region. (c) Overlay of E. coli KDPGal (cyan) aldolase complex with our previous model of the E. coli KDPG (violet) aldolase-substrate complex. Shown in stick are key residues, with oxygen and nitrogen atoms colored red and blue, respectively, in both, and carbon atoms colored yellow in KDPGal aldolase and green in KDPG aldolase. For KDPGal aldolase the residues shown are the same as in A (but not labeled). For KDPG aldolase, residues R49, V20, E45, K133, T161, G158, and S184 are shown. The side chain of R49 of E. coli KDPG aldolase overlaps with the side chain of R14 of E. coli KDPGal aldolase. The difference in main chain position is unlikely to be significant as the KDPG aldolase from T. maritime has R14 in the same position as E. coli KDPGal.

Scheme 1

Aldol addition catalyzed by KDPG and KDPGal aldolases.

Scheme 2

Preparative scale addition of pyruvate and pyridine carboxaldehyde. Reagents: (a) KDPGal aldolase; (b) HCl, EtSH.