Aggregation Interface and Rigid Spots Sustain the Stable Framework of a Thermophilic N-Demethylase

Enzymes from thermophilic microorganisms usually show high thermostability, which is of great potential in industrial application; to understand the structural logic of these enzymes is helpful for the construction of robust biocatalysts. In this study, based on the crystal structure of an N-demethylase-TrSOX-with outstanding thermostability from Thermomicrobium roseum, substitutions were introduced on the aggregation interface and rigid spots to reduce the aggregation ratio and the rigidity. Four substitutions on the aggregation interface-V162S, M308S, F170S, and V306S-considerably reduced the thermostability and slightly enhanced the catalytic efficiency. In addition, the thermostable framework was considerably disrupted in several multiple P -> G substitutions in several local motifs (P129G/P134G, P237G/P259G, and P259G/P276G). These structural fluctuations were in good accordance with whole-structure or partial root-mean-square deviation, radius of gyration H-bonds, and solvent-accessible surface area values in molecular dynamics simulation. Furthermore, these key spots were introduced into an unstable homolog from Bacillus sp., resulting in a dramatical increase in the half-life at 60 degrees C from <10 to 1440 min. These results could help understand the natural stable framework of thermophilic enzymes, which could be references for the construction of robust enzymes in industrial applications.