A geometry-guided computational framework for predicting functional immobilization orientations of plant peroxidases...

Enzyme immobilization depends not only on support chemistry, but also on the spatial orientation adopted by the protein after attachment. This study presents a computational framework guided by molecular geometry to screen favorable immobilization orientations of plant peroxidases on modified surfaces. Horseradish peroxidase, soybean peroxidase, and royal palm tree peroxidase were analyzed as three distinct case studies. Surface exposed anchoring residues were identified from experimental structures, and rigid body orientation sampling generated enzyme and surface configurations. Each configuration was evaluated using a functional orientation score that combined heme accessibility, catalytic channel exposure, anchoring site proximity, protein and surface separation, and steric feasibility. Three support chemistries, carboxylated, hydroxylated, and aminated, were assessed, producing nine enzyme and surface combinations. Among the three simplified surface chemistries evaluated, the hydroxylated neutral model produced the highest composite compatibility score for each of the three peroxidases. The preferred surface therefore represented three of the nine evaluated combinations, while the remaining six showed lower overall compatibility. Comparison of mean and maximum scores indicated that the best orientation did not always represent the most stable orientation ensemble, highlighting the need to consider score dispersion and sensitivity. Reactive residue mapping also identified enzyme specific anchoring regions capable of preserving catalytic channel exposure and limiting heme occlusion. Overall, the workflow integrates structural accessibility, orientation quality, and surface compatibility within an interpretable screening strategy. It provides a low cost route for prioritizing immobilization conditions before testing and can be extended to other enzymes whose catalytic performance depends on maintaining access to buried active sites after attachment.