4.2. Rhapsody and in Silico Saturation Mutagenesis Analysis

The Rhapsody tool [24] was used for automated scanning of all residue substitutions in 15LOX-2 to predict the functional consequences of single amino acid variants (SAV). A random forest-based classifier was trained on an integrated dataset of 20,854 missense mutations functionally characterized to date. For each training sample, eight features incorporating the effects of structural dynamics and sequence-based (co)evolution properties were calculated using ProDy [58], Evol [58], and PolyPhen-2 [62]. The 15LOX-2 structure (PDB id: 4NRE [20]) was used as input. Residue-averaged scores evaluated by Rhapsody, PolyPhen-2, and EVmutation [25] were examined for consolidation of the results.