Insertion of a Calcium-Responsive β‑Roll Domain into a Thermostable Alcohol Dehydrogenase Enables Tunable Control over Cofactor Selectivity

The RTX domains found in some secreted proteins fold into the β-roll secondary structure motif upon calcium binding, which enables folding to be localized extracellularly. We inserted an RTX domain from the adenylate cyclase of Bordetella pertussis into a loop near the catalytic active site of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus. The resultant chimera, β-AdhD, gained the calcium-binding ability of the β-roll, retained the thermostable activity of AdhD, and exhibited reduced overall alcohol dehydrogenase activity. However, the addition of calcium to β-AdhD preferentially inhibited NAD+-dependent activity in comparison to NADP+-dependent activity. Calcium was found to be a competitive inhibitor of AdhD, and the addition of the RTX domain introduced calcium-dependent noncompetitive inhibition to β-AdhD affecting NAD+-dependent activity. Thus, the insertion of an intrinsically disordered calcium-binding domain into a key loop in a cofactor-dependent enzyme results in an enzyme with tunable cofactor selectivity, reminiscent of a calcium-controlled cofactor selectivity rheostat switch.