Figure 4.

Identification of residues required for optimal NAD(P)⁺ hydrolysis by Tne2_tox. A, molecular docking places NAD⁺ in the predicted active-site pocket of Tne2_tox. Phe-330, Arg-343, Lys-353, and Gln-413, which are conserved among Tne2 orthologous proteins and line the NAD⁺-binding pocket, are indicated. B, growth of E. coli cells expressing a vector control (Ctrl), Tne2_tox, or the indicated site-specific variants of Tne2_tox. C, SDS-PAGE analysis of the Tne2_tox–Tni2 complex and refolded Tne2_tox and the indicated site-specific mutants thereof. Tne2_tox and its variants possess an N-terminal hexahistidine tag, whereas Tni2 is untagged. Proteins were visualized using Coomassie Blue staining. D and E, hydrolysis rates of NAD⁺ (D) and NADP⁺ (E) by the purified proteins indicated in C. Error bars represent ± S.D. (n = 3). Asterisks indicate NAD(P)⁺ hydrolysis rates that are significantly higher than the Tni2-inhibited control.