In Silico Study of Potential Binding Sites of the Family GH126 Enzyme CPF_2247 from Clostridium perfringens Using Structural Comparison and Molecular Docking Methods

The family GH126 represents a potential fourth, but still non-confirmed α-amylase family in CAZy with the founding, partially characterized member, the assumed amylolytic enzyme CPF_2247 from Clostridium perfringens. Proteins of this family adopt an (α/α)6-barrel domain, structurally distinct from the rather more complex domain arrangement of families GH13, GH57, and GH119. Interestingly, GH126 exhibits structural similarity, including sharing potential functionally important residues with inverting β-glucanases from GH8 and GH48 (clan GH-M); this fact has prompted previous bioinformatics analyses. In the present study, two GH126 members with experimentally determined tertiary structure—the CPF_2247 and the exopolysaccharide-specific hydrolase PssZ from Listeria monocytogenes—were compared with seven GH8 and ten GH48 enzyme-substrate complexes. Family GH126 enzymes display a wide, open binding cleft, with a central tunnel-like cavity along the barrel axis, distinct from the narrow cleft in GH8 and the tunnel-shaped site in GH48. Conserved residues involved in substrate binding and catalysis of GH8 and GH48 were identified in GH126. Molecular docking with α-glucans using the CPF_2247 confirmed predicted binding at the potential active site and revealed also eventual additional binding sites. Targeted docking showed the strongest interactions for acarbose and maltoheptaose, particularly involving a GH126 unique α11-α12 loop in the assumed amylolytic enzyme CPF_2247.