© 2019 Tak et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD in the absence of any exogenous cofactor, thus classifying it as a -NAD glycohydrolase. However, TNT lacks sequence similarity with other NAD hydrolyzing enzymes and lacks the essential motifs involved in NAD binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP as fast as NAD but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD-binding pocket and four glycine residues that form a cradle directly below the NAD-binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD-binding site revealed that Phe 727 , Arg 757 , and Arg 780 are essential for NAD hydrolysis by TNT. These results identify the NAD-binding site of TNT. Our findings also show that TNT is an NAD glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.