cs7b03809_si_001.pdf (1.65 MB)
Insertion of a Calcium-Responsive β‑Roll Domain into a Thermostable Alcohol Dehydrogenase Enables Tunable Control over Cofactor Selectivity
journal contribution
posted on 2018-01-02, 00:00 authored by Walaa Abdallah, Kusum Solanki, Scott BantaThe 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.