Fig. 3. NAD⁺ cleavage activity is intrinsic to the N-terminal sirtuin-like domain of ThsA.

a Structural alignment of the ThsA N-terminal domain (blue) with a sirtuin deacetylase, T. maritima CobB (PDB ID: 2H2F; gray) bound to a co-substrate, NAD⁺ (green). The essential zinc ion in the CobB structure is represented as a gray sphere. b Close-up view of the NAD⁺ binding site. Asn112 and His152 of ThsA align with sirtuin residues interacting with NAD⁺. c Sequence alignment of the N-terminal domain of ThsA with sirtuin proteins. Asn112 and His152 of ThsA are marked with asterisks. White character in red box indicates strict identity. Red character and blue frame represent similarity in a group and across groups, respectively. Alignment of the full-length amino acid sequences is presented in Supplementary Fig. 6. d Asn112 and His152 of the N-terminal sirtuin-like domain are critical for NAD⁺ hydrolysis of ThsA. N112A and H152A mutations of ThsA abolished NAD⁺ cleavage activity of ThsA. The graph for the wild-type ThsA (Fig. 1a) is shown again as a control for comparison. Data are normalized to the values measured at 0 min for each protein and presented as mean ± SEM for three independent experiments. Source data are provided as Source Data file.