Mechanism of Sirtuins

Avalos, J.L., Bever, K.M. and Wolberger, C. (2005) Mechanism of sirtuin inhibition by nicotinamide altering the NAD( + ) cosubstrate specificity of a Sir2 enzyme. Molecular Cell, 17 (6), 855-868. 

Despite the large amount of biochemical and structural studies of sirtuins in complex with various substrates, cofactors and reaction products, the catalytic mechanism of this class of enzymes is still a matter of debate. SN -like [56] and SN -like [60] mechanisms have been inferred from structural studies but further biochemical and possibly structural studies will be required to clarify which mechanism is used by sirtuins. It should also be noted that another matter of debate concerns the mode of noncompetitive inhibition of sirtuins by the reaction product nicotinamide [62], various structural studies having highlighted different binding pockets for this molecule [63, 64]. 

Resveratrol (Fig. 23), a stilbene found in many food sources, e.g., peanuts and red wine, is assumed to have multiple benefits on human health. Most attention has been received by the so-called French paradox , the low occurrence of cardiovacular disease in populations living on a diet high in saturated fats, but consuming red wine. The protective effect of red wine is attributed to its proanthocyanidin and resveratrol contents. Possible mechanisms disscussed are the inhibition of oxidation of LDL cholesterol and platelet aggregation. Resveratrol may also increase longevity by activation of sirtuins, NAD+-dependent protein deacetylases involved in aging, which respond to oxidative stress and are induced by a low-calory diet. Resveratrol mimics the effects of a low-calory diet and extends the... 

Although the localisation patterns of some of the Sirtuins (class III HDACs) and their unique NAD-dependent deacetylation mechanism are known [58], less is understood about their functions and targets when compared to other HDACs [59]. The field of small molecule Sirtuin modulators is also correspondingly less advanced, because this alternative mechanism renders the zinc-dependent... 

So far 18 different members of HDACs have been discovered in humans and classified into four classes based on their homology to yeast histone deacetylases [33]. Class I includes four different subtypes (HDACl, 2, 3, 8), class II contains six subtypes tvhich are divided into two subclasses class Ila with subtypes HDAC4, 5, 7, 9 and class Ilb with HDAC6, 10. Class I and class II HDAC share significant structural homology, especially within the highly conserved catalytic domains. HDACs 6 and 10 are unique as they have two catalytic domains. HDACll is referred to as class IV. While the activity of class I, II and IV HDACs depends on a zinc based catalysis mechanism, the class III enzymes, also called sirtuins, require nicotinamide adenine dinucleotide as a cofactor for their catalysis. 

The equilibrium of reversible histone lysine acetylation is maintained by histone deacetylases (H D ACs) on one hand and histone acetyltransferases on the other hand. Human histone deacetylases can be separated into four classes [15]. HDACs of class I, II and IV are zinc-dependent amidohydrolases, whereas class III HDACs, also referred to as sirtuins, have a mechanism that is dependent on NAD [16]. As histone deacetylases have been widely studied, it is not surprising that there are also a large number of assays existing that have helped to characterize modulators of these enzymes and subsequently the enzymes themselves. 

Compared to the zinc-dependent HDACs, the sirtuins act by a very different mechanism and require NAD+ as a cofactor. Unsurprisingly, they show no sequence similarity with the other HDACs and are structurally very distinct [97]. The size of most sirtuins (Sirt2 to Sirt7) varies from 310 to 400 amino acid residues, while Sirtl is larger (747 residues). Multiple crystal structures of eukaryotic and prokaryotic sirtuin proteins have been reported, which either are apo-forms or include ligands such as NAD+ derivatives, W-acetylated lysine substrates, and/or other small molecules [98-110]. These data have shed much light on the mode of action of this enzyme class. 

As illustrated in Fig. 9, sirtuins convert one equivalent of NAD+ to nicotinamide and 2/-0-acetyl-ADP-ribose (2 -OAADPr) to deacetylate an Ve-acetyl lysine group [111]. This mechanism requires a conformational change of NAD+ resulting in weakening of the Cl -N bond, which is induced upon binding of the substrate to the enzyme [109, 112]. A nucleophilic substitution at the anomeric... 

Several structurally diverse sirtuin inhibitors have been reported, some of which are illustrated in Fig. 10. Nicotinamide is a product of NAD+ degradation that occurs during sirtuin-mediated catalytic process. Its inhibitory function at high concentrations is a result of a reaction with the ribosyl oxycarbenium intermediate formed as part of the mechanism, thus reversing the catalytic process and preventing deacetylation. Sirtinol 26 and salermide 27 [116], cambinol 28 [117], the tenovins 29 [118], and splitomycin 30 all show moderate inhibitory activity in the micromolar range. 

Recently, in an approach to explain the diverse actions of polyphenols, Howitz et al. suggested that the antiproliferative and oncosuppressive properties of resveratrol might be due to a mechanism that mimics caloric restriction and lifespan extension, and involves the sirtuin (SIRT) family of nicotinamide adenine dinucleotide (NAD) -dependent acety-lases (Howitz et al. 2003). More specifically, resveratrol was found to directly interact with SIRTl deacetylase, resulting in decreased acetylation of p53, increased DNA stability, and finally cell survival. Redox formation was implicated in the inhibition of histone deacetylase (HDAC) activity, leading to a chronic inflammatory-like response (Rahman et al. 2004). In this respect, resveratrol is a promising agent in the reversal of oxidative stress and rescue of mutant phenotypes. 

Only very recendy, it was discovered that a class of histone deacetylases, the simiins, is dependent on NAD. Sirtuins deacetylate proteins and concomitantly cleave NAD The reaction mechanism appears to be unique for ADP-ribosyl transfers. ... 

Histone deacetylases (HDACs, EC number 3.5.1) remove acetyl groups from A -acetyl lysines by hydrolysis, both on histones and non-histone proteins, hence are more generally referred to as lysine deacetylases (KDACs). HDACs are grouped into four classes based on sequence homology and mechanism (Table 5.2). The first two classes, sometimes referred to as classical HDACs, are zinc-dependent and their activity is inhibited by hydroxamic acids, e.g. trichostatin A (TSA). The third class, referred to as Sirtuins, are NAD -dependent proteins and are not inhibited by TSA. The fourth class is also zinc-dependent, but is considered an atypical category based on low sequence homology to classes I and II. Class I and IV HDACs are mainly found in the nucleus and are expressed in many cell types, while the expression of class II HDACs, which are able to shuttle in and out of the nucleus, is tissue specific. Sirtuin localisation depends on the particular isoform (cytoplasm, mitochondria and nucleus). 

The proposed mechanism for the NAD -utilising HDACs (Sirtuins) is more complex, involving in the first step the formation of an O-glycosyl iminoyl ether and release of nicotinamide (Figure 5.10b). The iminoyl ether is then hydrolysed by a molecule of water, assisted anchimerically by the 2 -OH group of the ribosyl moiety, to give 2-O-acetyl ADP-ribose and the deacetylated lysine. 

The human genome contains 18 HDACs that are classified according to their catalytic mechanism. The focus of this chapter is the eleven zinc-dependent HDACs 1-11, which contain a zinc cation as the active site catalyst. In addition, there are seven sirtuins, SIRTs 1-7, which instead employ the cofactor NAD for amide bond hydrolysis. The zinc-dependent HDACs are further subdivided into elass I (HDACs 1, 2, 3 and 8), class Ila (HDACs 4, 5, 7 and 9), class Ilb (HDACs 6 and 10) and class IV (HDAC 11) based on sequence homology and cellular loealization. The class I HDACs are ubiquitously expressed and primarily located in the cell nucleus, where... 

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