Structure of a rabbit muscle fructose-1,6-bisphosphate aldolase A dimer variant

Abstract

Fructose-1,6-bisphosphate aldolase (aldolase) is an essential enzyme in glycolysis and gluconeogenesis. In addition to this primary function, aldolase is also known to bind to a variety of other proteins, a property that may allow it to perform 'moonlighting' roles in the cell. Although monomeric and dimeric aldolases possess full catalytic activity, the enzyme occurs as an unusually stable tetramer, suggesting a possible link between the oligomeric state and these noncatalytic cellular roles. Here, the first high-resolution X-ray crystal structure of rabbit muscle D128V aldolase, a dimeric form of aldolase mimicking the clinically important D128G mutation in humans associated with hemolytic anemia, is presented. The structure of the dimer was determined to 1.7 angstroms resolution with the product DHAP bound in the active site. The turnover of substrate to produce the product ligand demonstrates the retention of catalytic activity by the dimeric aldolase. The D128V mutation causes aldolase to lose intermolecular contacts with the neighboring subunit at one of the two interfaces of the tetramer. The tertiary structure of the dimer does not significantly differ from the structure of half of the tetramer. Analytical ultracentrifugation confirms the occurrence of the enzyme as a dimer in solution. The highly stable structure of aldolase with an independent active site is consistent with a model in which aldolase has evolved as a multimeric scaffold to perform other noncatalytic functions.

Figure 1

Rabbit muscle fructose-1,6-bisphophate aldolase tetramer (Blom & Sygusch, 1997; PDB code 1ado). Each monomer is colored separately, with the A and B interfaces indicated by arrows as described previously (Beernink & Tolan, 1994 ▶). The sites of the D128V substitutions are indicated by red spheres on interface B and the C- and N-termini are labeled for monomer A.

Figure 2

Stereoview of the site of substitution in D128V aldolase. The residues flanking the site of substitution are shown with the corresponding 2F _o − F _c electron-density map (blue cages) contoured at 1σ.

Figure 3

Overlay of D128V aldolase (cyan) and wild-type aldolase (yellow). The C^α traces are oriented as in Fig. 1 ▶.

Figure 4

Sedimentation-equilibrium analytical ultracentrifugation of D128V aldolase under crystallization conditions. The sedimentation-equilibrium trace using 20 µM protein at 291 K and 8000 rev min⁻¹ (monitored by absorbance at 280 nm) is shown in the lower panel. The solid line depicts the best fit to the data (open circles) performed as described in §2.4. Residual errors are presented in the upper panel.

Figure 5

The B interface of wild-type aldolase A. Depiction of the hydrogen bonds (dashed lines) made with Asp128 at the B interface. The coloring scheme is identical to that of Fig. 1 ▶ for the ribbons, with a ball-and-stick representation in yellow for one monomer and in orange for the second monomer.

Figure 6

Stereoview of the active site of fructose 1,6-bisphosphate. (a) DHAP and a sulfate ion depicted in ball-and-stick representation along with conserved active-site residues in D128V aldolase. A 1σ electron-density map is shown (violet cages). (b) Superposition of the active-site residues of D128V aldolase in red with DHAP and sulfate ion in yellow and the residues of the wild-type DHAP-bound structure (Blom & Sygusch, 1997; PDB code 1ado) in green. Hydrogen bonds are shown in black for D128V aldolase and in green for the wild-type structure.