Two Novel Mutant Human Adenylosuccinate Lyases (ASLs) Associated with Autism and Characterization of the Equivalent Mutant Bacillus subtilis ASL

An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a “humanized” normal B. subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). Vmax for conversion of adenylosuccinate to AMP and fumarate is 0.57 μmol/min/mg for I62E/D65R, 0.064 μmol/min/mg for I62D/D65R, 0.27 μmol/min/mg for I62E/D65R/D69E, and 0.069 μmol/min/mg for I62D/D65R/D69E. The Km for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 °C. The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp62 causes the most drastic effect on ASL function, whereas the Glu69 mutation produces only modest change. These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.