The structure and function of the most prevalent mutant form of aldolase B associated with hereditary fructose intolerance.

摘要

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder affecting one in 20,000 individuals. HFI is caused by mutations in the gene encoding aldolase B, a tetrameric enzyme central to the gluconeogenic and fructose metabolism pathways. The most common mutation is an Ala → Pro substitution at position 149 (A149P), which accounts for 53% of the HFI alleles identified worldwide. Elucidation of the structural and functional characteristics of the aldolase B A149P substitution (AP-aldolase) required expression as a glutathione S-transferase fusion protein. The fusion protein promoted stability and facilitated purification of AP-aldolase. Circular dichroism spectroscopy on purified AP-aldolase shows secondary and tertiary structural transitions (T_1/2) at 45°C and 33°C, respectively. Kinetic investigations reveal that the residual activity of AP-aldolase is sensitive to temperature, having 15% of wild-type activity at 10°C, but only about 0.5% activity at 30°C, with T_1/2 = 25°C. In contrast, changes in the K_m, values compared to wild-type toward the substrates fructose-1,6-bisphosphate, fructose-1-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate are temperature-independent, and suggest a perturbation at the C(1)-phosphate binding site. Gel-filtration chromatography and zonal sedimentation experiments show that the quaternary structure of AP-aldolase is disrupted at all temperatures, being mostly dimeric. The A149P substitution is not located at a subunit interface in the aldolase structure. To further clarify the structural perturbations, crystals of AP-aldolase were grown at two temperatures (4 and 18°C), and the structure solved at 3.0 Å resolution by X-ray crystallography, using the wild-type structure as the phasing model. The structure reveals that the single residue substitution causes significant perturbation and molecular disorder at the site of mutation (residues 148–158), which is propagated onto three adjacent β-strand and loop regions (residues 110–128, 189–200, 234–243). Disorder in the 110–128 loop region provides an explanation for the disrupted quaternary structure. Greater structural perturbation is observed in the structure determined at 18°C. These results are discussed in the context of HFI, and the limitations of homology modeling in predicting perturbations of protein structure and function.