Kinetic and Structural Basis for Acyl-Group Selectivity and NAD⁺ Dependence in Sirtuin-Catalyzed Deacylation

Abstract

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.

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

“…These findings are in keeping with the presence of nuclear metabolic territories as recently predicted (41,42) and prompted us to investigate whether nuclear NADH distribution could be influenced by SIRT1. Importantly, the K m of SIRT1 for NAD + seems to fall into the physiological range of NAD + bioavailability in the nucleus (43).…”

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH

“…Molecular Modeling; Structural Basis for Deacylation and NADH Inhibition-The findings that SIRT1-3 and SIRT6 can remove long-chain acyl groups prompted us to investigate the molecular basis for accommodation of long acyl-amide substrates in the active sites of sirtuins 1-3 and 6. The active sites of SIRT2 and -6 have been recognized to contain a hydrophobic pocket accommodating the long acyl chain of a myristoylated peptide (18,68) that is distinct from the NAD ϩ cofactor binding site (75). Based on published x-ray crystal structures, we analyzed the hydrophobicity of the active sites of SIRT1-3 and SIRT6 and found that a hydrophobic pocket was also present in SIRT1 and SIRT3 (Fig.…”

Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins

“…Importantly, of the seven sirtuins encoded in the human genome, only Sirt1, -2, and -3 have a robust deacetylase activity. The other sirtuins have preferences for longer acyl chains (56,57).…”