Structural determinants of the thermostability of d-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii.

Abstract

d-Amino acid oxidase from the thermophilic fungus Rasamsonia emersonii strain YA (ReDAAO) exhibits high thermostability. To understand the structural basis for this high stability, we isolated thermolabile variants of ReDAAO with a single amino acid substitution (L134P, K203E, C230S, V275G, and V305L), whose T₅₀ (the temperature at which 50 % of the initial enzyme activity was retained) values were 12-18 °C lower than that of the wild-type. The L134P substitution in a flexible protein surface loop caused the most severe destabilization, likely due to increased loop flexibility through hydrogen bond disruption. The other substitutions affected stability by impairing distinct structural elements: K203E might disrupt an amino acid interaction network involved in both flavin adenine dinucleotide binding and subunit interactions, C230S might eliminate the unique disulfide bond that likely fixes a long α-helix involved in subunit interactions, and V275G and V305L might perturb critical interactions at subunit interfaces, with V305L also potentially affecting the subunit structure. Notably, the thermostabilization conferred by the disulfide bond and the interaction network involving K203 were unique to thermophilic fungal DAAOs. These findings revealed multiple distinct mechanisms of thermostabilization in ReDAAO, providing valuable insights for engineering flavoenzymes with improved thermostability.