Characterization of E121K mutation of D-amino acid oxidase – Insights into mechanisms leading to amyotrophic lateral sclerosis

D-amino acid oxidase (DAO) maintains the intracellular d-serine level which modulates the activity of the N-methyl-d-aspartate receptor and its dysfunction has been linked to several neurodegenerative disorders. In targeted next-generation sequencing study by our group, E121K mutation in DAO was associated with amyotrophic lateral sclerosis (ALS) in patients from India. However, variations in molecular mechanisms caused by this mutation which leads to ALS have not been studied. Hence, we carried out comparative biophysical characterization and assay studies of the wildtype- and mutant E121K-DAO. We observed that the purified E121K-DAO was inactive and exhibited a lower affinity for the FAD cofactor and benzoate inhibitor. Structural studies revealed that the E121K mutant has higher beta-sheet content, melting temperature, and oligomeric states compared to the wildtype. Kinetic study of aggregation of the variants using thioflavin-T confirmed that the E121K-DAO was more prone to aggregation. Microscopic visualization showed that the aggregation proceeds through an intermediate step involving the formation of fibrillar structures in the E121K mutant. Our results give insights into the underlying mechanisms leading to ALS pathogenesis.