Structure comparison and identification of a Sirtuin sequence motive indicating nicotinamide insensitive deacetylation activity.

<p>(<b>A</b>) Structure comparison of Sirt3 (red) and Sirt5 (blue), overlaid with a Sir2Tm/nicotinamide complex (grey). Nicotinamide and coupled residues (see below) in Sirt3 and Sirt5 are displayed as sticks and colored by atom type for Sirt5 and for nicotinamide. (<b>B</b>) Statistical coupling analysis scores (lower panel) identify residues apparently coevolving (score cutoff used: 1.5) with Sirt5-Arg105: Thr69 and Tyr102 (highlighted in panel A), Trp77, Arg217, and Trp222. Amino acids in other Sirtuin sequences and the corresponding Sirtuin class are shown on top of the scores. (<b>C</b>) Inhibition Sirt5-Arg105Leu by nicotinamide. Activities, determined using our mass spectrometry assay, were normalized against activity in absence of nicotinamide. Data for wildtype Sirt3 and Sirt5 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045098#pone-0045098-g002" target="_blank">Fig. 2A</a> are shown here for comparison. (<b>D</b>) Comparison of IC₅₀ values, determined using the mass spectrometry assay, for nicotinamide inhibition of class I and III Sirtuins and of the Sirt5-Arg105Leu variant. Error bars represent the standard error of the fit. NAM, nicotinamide.