Fig. 3. Crystal structures of CBADH_S and TBADH_S1 reveal mechanism of cofactor selectivity reversal.

a Cofactor binding site of CBADHS with NAD⁺ bound. The substitutions identified in CBADH_S allow for the binding of NAD⁺ but would prevent the binding of NADP⁺ by steric impediments and electrostatic repulsion with the side chains of D198 and Y199. Additionally, the stacked-ring interaction of the adenine moiety with Y218 observed in CBADH_WT cannot be established due to the Y218P substitution, possibly enabling a more flexible binding of the cofactor. b Cofactor binding site of TBADH_S1, with NAD⁺ placed at the same position as NADP⁺ in the structure of TBADH_WT. P200−P201 is modelled as a cis peptide bond. While TBADH_S1 can accommodate NAD⁺ in its cofactor binding pocket, the binding of NADP⁺ would be prevented by steric impediments caused by the side chains of S198 and K199. As in CBADH_S, the substitution of Y218 prevents the formation of a stacked-ring interaction with the adenine ring of the cofactor, possibly enabling a more flexible binding of NAD⁺.