(626d) Insertion of a Calcium-Binding Beta Roll Domain into a Thermostable Alcohol Dehydrogenase Enables Allosteric Control over Cofactor Specificity

The RTX domain from the adenylate cyclase toxin of Bordetella pertussis was cloned into thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus. The insertion was made in loop A, between the 124^th and 125^th amino acid of AdhD, a member of the aldo-keto reductase (AKR) superfamily. The fusion protein, β-AdhD, retained the calcium-binding ability of the RTX domain. The formation of the β-roll in β-AdhD was confirmed via Förster resonance energy transfer, where terbium, a calcium analog, showed a higher fluorescence emission in the fusion protein compared to the wild type enzyme. Additionally, the fusion retained the thermostability of AdhD, as well as its ability to oxidize alcohols. Although β-AdhD is less active than AdhD, it gained the ability to modulate activity upon calcium addition and interestingly, this was only seen in the presence of NAD⁺. Furthermore, calcium was determined to be a mixed inhibitor with β-AdhD with NAD⁺ while no clear inhibition was determined in the presence of NADP⁺. Therefore, not only does calcium modulate activity, but it also imparts cofactor preference in β-AdhD, where NAD⁺ is preferred in the absence of calcium and NADP⁺ is preferred in the presence of calcium. This work can be utilized in the development of multi-step catalytic cascades where activity can be controlled externally. Additionally, this approach is modular in that the insertion site, loop A, is found in all members of the AKR superfamily. Thus, we have demonstrated that by inserting a calcium-binding domain into a key catalytic loop in a cofactor-dependent enzyme, we have created an allosterically-regulated enzyme with tunable cofactor selectivity, reminiscent of a calcium-controlled cofactor selectivity rheostat switch.

Abdallah, W., Solanki, K., and Banta, S. “Insertion of a calcium-binding beta roll domain into a thermostable alcohol dehydrogenase enables allosteric control over cofactor specificity” Journal of Molecular Biology (Submitted).