Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD+ biosynthetic pathway and is required for SirT1 activity in vivo. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.

Sirtuins are NAD(+)-dependent enzymes universally present in all organisms, where they play central roles in regulating numerous biological processes. Although early studies showed that sirtuins deacetylated lysines in a reaction that consumes NAD(+), more recent studies have revealed that these enzymes can remove a variety of acyl-lysine modifications. The specificities for varied acyl modifications may thus underlie the distinct roles of the different sirtuins within a given organism. This review summarizes the structure, chemistry, and substrate specificity of sirtuins with a focus on how different sirtuins recognize distinct substrates and thus carry out specific functions.

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Lysine propionylation is a recently identified post-translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyllysine is slightly reduced to acetyl-lysine. These results serve to broaden our understanding of the newly identified propionyl-lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ϵ-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

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The core histones, H2A, H2B, H3 and H4, undergo post-translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono-, di- and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high-performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom-up liquid chromatography-mass spectrometric analysis. The deuteroacetylation of unmodified or mono-methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross-talk’ by correlating different PTMs on the same histone tail.