Background: Sirtuins regulate metabolism, genome maintenance, and stress responses. Results: Long-chain free fatty acids stimulate SIRT6 deacetylase, and sirtuins display distinct but overlapping specificity for diverse acylated peptides. Conclusion: SIRT6 is activated by biologically relevant fatty acids, and long-chain deacylation is a general feature of sirtuins. Significance: Discovery of endogenous, small-molecule activators of SIRT6 demonstrates the therapeutic potential of compounds that promote SIRT6 function.

SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD ؉ -dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early reports suggested that SIRT6 performs ADPribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces Oacetyl-ADP-ribose but at a rate ϳ1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2 -N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD ؉ -binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD ؉ in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD ؉ with relatively high affinity (K d ‫؍‬ 27 ؎ 1 M) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD ؉ induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD ؉ metabolite sensor.Sirtuins comprise an ancient and diverse family of nicotinamide adenine dinucleotide (NAD ϩ )-dependent protein deacetylases that are evolutionarily conserved from bacteria to eukaryotes (1). Unlike other classes of histone deacetylases, which utilize an active site Zn 2ϩ and involve direct attack of a water molecule on acetylated lysines, sirtuins transfer the acetyl group from the lysine side chain of a protein or peptide substrate to the co-factor NAD ϩ , generating nicotinamide, 2Ј-Oacetyl-ADP-ribose (OAADPr) 3 (2, 3), and a deacetylated substrate. This unique requirement of NAD ϩ suggests that sirtuins might act as sensors of the cellular metabolic state (4), relaying changes in cellular metabolism to reverse acetylation-mediated pathways, which include transcription, cell cycle progression, genome maintenance, apoptosis, and organism longevity. The founding member of the sirtuin family, yeast Sir2 (silent information regulator 2), has emerged as an important regulator in extending the life span of Saccharomyces cerevisiae (5). In other organisms, such as Caenorhabditis elegans and Drosophila me...

Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long-chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity.1 Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl-substrate chain length. Though the rate of nucleophilic attack of the 2′-hydroxyl on the C1′-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however for SIRT6, this step is no longer rate-limiting for long-chain substrates. Co-crystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2′-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl-substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed.

Summary Hepatic glucose production (HGP) maintains blood glucose levels during fasting but can also exacerbate diabetic hyperglycemia. HGP is dynamically controlled by a signaling/transcriptional network that regulates the expression/activity of gluconeogenic enzymes. A key mediator of gluconeogenic gene transcription is PGC-1α. PGC-1α’s activation of gluconeogenic gene expression is dependent upon its acetylation state, which is controlled by the acetyltransferase GCN5 and the deacetylase Sirt1. Nevertheless, whether other chromatin modifiers—particularly other sirtuins—can modulate PGC-1α acetylation is currently unknown. Herein we report that Sirt6 strongly controls PGC-1α acetylation. Surprisingly, Sirt6 induces PGC-1α acetylation and suppresses HGP. Sirt6 depletion decreases PGC-1α acetylation and promotes HGP. These acetylation effects are GCN5 dependent: Sirt6 interacts with and modifies GCN5, enhancing GCN5’s activity. Leprdb/Leprdb mice, an obese/diabetic animal model, exhibit reduced Sirt6 levels; ectopic re-expression suppresses gluconeogenic genes and normalizes glycemia. Activation of hepatic Sirt6 may therefore be therapeutically useful for treating insulin-resistant diabetes.