Canavan disease (CD) is a fatal, childhood neurological disorder with altered N-acetylaspartate (NAA) metabolism. This disease is caused by mutations in the gene that encodes the enzyme aspartoacylase (ASPA) in the brain. The deficiency of aspartoacylase activity leads to the hallmark symptoms of CD. Sixteen different ASPA clinical mutations have been examined for their biophysical properties. Each of these recombinant mutant enzymes was found to have measureable catalytic activity, ranging from 0.3% to 35% of the native enzyme. Many of these mutants show either decreased thermal stability or decreased conformational stability. These results suggest that the loss of catalytic activity of ASPA is at least partially a consequence of decreased protein stability.
To study the possible structural defects caused by non-conservative amino acid changes in ASPA, four aspartoacylase mutations have been structurally characterized. The mutant enzymes each have similar overall structures to the native ASPA enzyme, but with varying degrees of alterations that offer explanations for the respective loss of catalytic activity. The loss of van der Waals contacts in the F295S mutation, and the loss of both hydrophobic and hydrogen bonding interactions in the Y231C mutation lead to a local collapse of the hydrophobic core structure contributing to a decrease in protein stability. The E285A mutant structure shows that loss of hydrogen bond interactions with the side chain carboxyl of Glu285 disturbs the active site architecture, leading to altered substrate binding and diminished enzymatic activity. Since many of the ASPA clinical missense mutations were found to have compromised stability, screening of a small molecule library was performed to identify compounds that can potentially bind and stabilize these defective enzymes. Three compounds (Glutathione, Patulin, and Ellagic acid) show significant protection against thermal denaturation of E285A, but they have negligible effect on the native enzyme. Optimization of these compounds, guided by kinetics and structural studies, could lead to drugs that have the potential to reverse the effect of these mutations in Canavan patients.
N-acetylaspartate is an enigmatic metabolite whose biological function is yet to be completely defined. In order to understand the physiological role of NAA in the brain and to find a treatment therapy for CD, the NAA biosynthetic enzyme aspartate N-acetyltransferase (AspNAT) was selected for examination. A codon optimized gene of AspNAT (NAT8L) was cloned with different fusion tags to enhance the solubility. The thioredoxin-tagged AspNAT was found to express as a soluble protein with low yield. This enzyme construct was partially purified using Ni affinity chromatography and was found to have N-acetyltransferase activity. Further optimizations are being carried out to obtain sufficient enzyme for biochemical characterization.