Preparation of model

The model structure of Sfamy R64 and mutants were prepared by homology modeling method using MODELLER 9.15,21,22 as reported previously.23 The selection for the template is a crucial step in homology modeling. The structure of A. niger α-amylase with PDB ID 2GUY was used as a template for all models based on the sequence similarity (71% homology) and the structure quality (1.59 Å). The model mutants of Sfamy R64 were built by changing and inserting some amino acid to mimic the positive control. The model structures were evaluated by the Ramachandran plot using PROCHECK and the Z-score using ProSA-web.24,25 The calcium ion (Ca²⁺) was added to all models located in the A/B domain as a cofactor. The structure of A. niger α-amylase with PDB ID 2GVY was used as a positive control. Maltose which bound to the C domain of A. niger α-amylase, or SBS, was used in all model systems studied by MD simulation.

Table 1 shows the five MD systems prepared in this study. The cysteine-type and the protonation state of histidine were manually annotated to their chemical environment. The parameters of the substrate (maltose) were calculated using the AM1-BCC method by antechamber program.26 The solvent system of a box TIP3P water was added to the solute with the least distance 10 Å between protein and the edge of the box. The sodium ion was used to neutralize the system.

The list of the MD system

Abbreviations: MT1, mutant 1; MT2, mutant 2; MT3, mutant 3.