Fig. 5: Viability assays and mutagenesis studies.

a Viability assays of E coli cells (BL21-AI and MG1665 strains) ectopically expressing indicated DSR2 variants (pACYCDuet) and SPR TTP (pBAD). The expression was either induced (1 mM IPTG and 0.2% L-Ara) or uninduced. b Growth curves of E. coli MG1655 expressing indicated DSR2 variants and SPR TTP. The expression was induced by adding 1 mM IPTG and 0.2% L-Ara when the bacteria were grown to OD₆₀₀ between 0.2 and 0.3. Each curve represents the mean ± SD of n = 3 independent biological replicates. c Intracellular NAD⁺ concentration of bacteria expressing indicated DSR2 variants and SPR TTP. Left, the expressions were induced; middle, the expressions were uninduced. Right, NAD⁺ level of bacteria co-expressing DSR2 and SPR TTP when induced at different ratio between IPTG to L-Ara (indicated on top). NAD⁺ measurements were taken every 20 min, and each curve represents the mean ± SD of n = 3 independent biological replicates. d NADase activity of DSR2-TTP complexes harboring indicated mutations at five key regions: the DSR2 CTD-SIR2 interface (red background), the SIR2-SIR2 interface (yellow background), the DSR2-TTP interface on TTP (blue background), the DSR2-TTP interface on DSR2 (gray background) and the DSR2-DSR2 interface (green background). Data is presented as mean ± SD from n = 3 independent biological replicates. e Cryo-EM structure of the DSR2-TTP complex illustrating five key regions for mutagenesis; residues involved in mutagenesis are shown with spheres and colored as in (d). While the DSR2-TTP complex is colored gray, the top right DSR2 is highlighted cyan and the top right TTP is highlighted dark blue. f A working model for activation mechanism of DSR2 system.