Fig. 6: MD simulations revealed the NAD⁺ hydrolysis mechanism of the Sir2 ___domain of GsSir2.

a The most representative structures are SPARSA-NAD⁺ and SPARSA-gRNA/tDNA/NAD⁺. Sir2, APAZ, PIWI, MID, and gRNA/tDNA are represented by cyan, green, magenta, yellow and orange cartoons, respectively. NAD⁺ is colored in orange (SPARSA) or purple (SPARSA-gRNA/tDNA). NAD⁺ and key residues are shown in sticks. The water molecules are represented by red spheres. b The number of water molecules around the C1’ atom of NAD⁺ within a cutoff of 3.5 Å. Data are represented as violin plots with box and whisker (lower and upper hinges correspond to the first and third quartiles, mean values are presented by circle within the box, whiskers extend from the hinge to the largest/smallest value). The maximal, central, and minimal values observed are as follows: For SPARSA, the values were 3, 0, and 0; for SPARSA-gRNA/tDNA, they were 5, 2, and 0; for N142A, the values were 5, 0, and 0; and for H186A, they were 4, 1, and 0. The statistical significance was analyzed by using one-way ANOVA followed by Bartlett test. A p value of less than 0.05 was considered statistically significant. c The NADase activity of WT SPARSA-gRNA/tDNA and the Y89A, R190A and Y89A & R190A SPARSA-gRNA/tDNA mutations. Each value is the mean ± SD and from more than three replicates. d A remarkable conformation of loop1 (¹⁸⁶HGD¹⁸⁸) caused by the hydrogen bond interaction between H186 and H163. e, f The distance between the Nε atom of H186 and the O atom of H186 and the distance between loop1 and C1’ in SPARSA-NAD⁺ (e), SPARSA-gRNA/tDNA/NAD⁺ (f), H186A (g) and N142A (h). i Schematic of the NAD⁺ hydrolysis mechanism of the Sir2 ___domain. The hydrogen bond interaction between H186 and H163 may allow the presence of water molecules for an α-face nucleophilic attack on C1’.