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. 2021 Mar 12;12:1631. doi: 10.1038/s41467-021-21307-z

Fig. 4. Crystal structure of AfNADase in complex with reaction products or the non-hydrolysable NAD+ analogue BAD.

Fig. 4

A Complex of AfNADase and the reaction products Nam and ADPR. The protein is shown as a cartoon embedded into a transparent surface map. The palm domain is coloured blue and the reaction products Nam and ADPR are displayed in stick representation. B Calculated mFo-DFc POLDER electron density map contoured at 3.0 σ to confirm the binding of ADPR and nicotinamide. C Residues of AfNADase involved in the interaction with the reaction products Nam and ADPR are shown in stick representation. The water molecule participating in the interaction is labelled WI. The dotted lines represent hydrogen bonds between the protein, water molecule and the reaction products. D In comparison with NAD+ the nitrogen in the pyridine rings has been substituted with a carbon atom in the non-hydrolysable analogue BAD. E Complex of AfNADase and the substrate analogue BAD. The protein is shown as a cartoon embedded into a transparent surface map. The palm domain is coloured blue and BAD is shown in stick representation. F Calculated mFo-DFc POLDER electron density map contoured at 3.0 σ to confirm the binding of BAD. G Residues of AfNADase involved in the interaction with BAD are shown in stick representation. The water molecules participating in the interaction are labelled WI and WII. The dotted lines represent hydrogen bonds between the protein, water molecules and the substrate analogue. H Proposed reaction mechanism of AfNADase-mediated NAD+ hydrolysis. In an SN2-like reaction mechanism, water molecule I is hydrogen-bonded to the in-ring oxygen of the proximal ribose, the nicotinamide moiety, the phosphodiester backbone and R129. This acts as a bridge to stabilize a positive change on the in-ring oxygen. The nucleophilic attack is further prepared by Q194, which interacts with the 2” OH of the proximal ribose and induces a δ-negative charge. The attack on the C1’ position from water II coincides with the nicotinamide leaving the active site and the formation of an oxonium intermediate. I activity of recombinant AfNADase WT and active site mutants R129A, F130A, F137A, Q194A and Q194K measured by the fluorometric assay using ɛNAD, n = 3. The experiment was performed independently three times with similar results. Source data are provided as a Source Data file.