Structure-function insights into the role of D-amino acid oxidase mutations implicated in amyotrophic lateral sclerosis.

Abstract

The D-amino acid oxidase (DAO) protein is a flavoenzyme that metabolizes certain D-amino acids and maintains motoneuron function by regulating D-serine levels and N-methyl-D-aspartate-receptor activation in the brain and spinal cord. Mutations in DAO are associated with Amyotrophic Lateral Sclerosis (ALS). We aim to understand the pathogenic basis of known DAO mutations through biophysical and biochemical assays, and X-ray crystallography. The recombinant protein of the wild-type and Site Directed Mutagenesis generated mutants were expressed via IPTG induction in E.coli (BL21), purified using affinity (Nickel-NTA) and Size Exclusion Chromatography, and used for biophysical, biochemical and crystallographic experiments. Oxidase enzyme activity assays with D-serine and D-alanine as substrates revealed that the mutations affected the efficiency of the enzyme. To determine the reason behind this, Circular-Dichroism spectroscopy, Thermal Fluorimetry, Differential Scanning Calorimetry and Tryptophan fluorescence profile of the mutants was analyzed to ascertain whether the changes in enzyme activity are attributable to changes in secondary/ tertiary structure of the proteins. The mutants retained their overall secondary and tertiary structure but the melting temperatures were altered to some extent. Ligand binding assays were also performed with FAD (cofactor) and Benzoate (inhibitor) to determine the effect of mutations on the binding affinity. The assays revealed that the binding of mutants with both FAD and benzoate is hampered due to the point-mutations. The X-ray crystallographic data will be combined with the biophysical information obtained, to determine the structural impact of the mutations on the functioning of the protein. However, it can be concluded that the reduced enzymatic activity may hinder the degradation of D-serine which may accumulate and alter the homoeostasis of the motoneurons, consequently contributing to the pathogenic effect of the mutations.