Improved homology model of cyclohexanone monooxygenase from Acinetobacter calcoaceticus based on multiple templates

•We build an improved model of cyclohexanone monooxygenase from Acinetobacter sp.•The developed homology model includes the nicotinamide cofactor for the first time.•We examine the enzymatic mechanism describing key residues during the reaction. A new homology model of cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus is derived based on multiple templates, and in particular the crystal structure of CHMO from Rhodococcus sp. The derived model was fully evaluated, showing that the quality of the new structure was improved over previous models. Critically, the nicotinamide cofactor is included in the model for the first time. Analysis of several molecular dynamics snapshots of intermediates in the enzymatic mechanism led to a description of key residues for cofactor binding and intermediate stabilization during the reaction, in particular Arg327 and the well known conserved motif (FxGxxxHxxxW) in Baeyer–Villiger monooxygenases, in excellent agreement with known experimental and computational data.