Update

TRENDS in Biochemical Sciences

Vol.30 No.9 September 2005

Research Focus

Vitamin B3 and sirtuin function

John M. Denu
University of Wisconsin Medical School, Department of Biomolecular Chemistry, 1300 University Avenue, Madison, WI 53706, USA

Sirtuins are NADC-dependent protein deacetylases that

are involved in transcriptional regulation, metabolism,
apoptosis, differentiation and ageing. These unique
enzymes are inhibited by nicotinamide, which is a
form of vitamin B3. Recent studies have uncovered the
molecular basis for nicotinamide inhibition, and provided the framework to understand the physiological
processes mediated by sirtuins and to develop strategies to modulate their cellular activity.
Introduction
Vitamin B3 (niacin) is a water-soluble vitamin that occurs
in two forms: nicotinamide and its acid form, nicotinic
acid. Cellular niacin can be obtained from two distinct
routes. Niacin can be made in the body from the precursor
tryptophan, which is obtained from foods rich in this
amino acid. Alternatively, niacin can be acquired directly
from the diet. Historically, adequate dietary niacin is
known to prevent the disease pellagra. More recently, the
health benefits of niacin have been shown to be far more
extensive than previously appreciated (http://www.umm.
edu/altmed/ConsSupplements/VitaminB3Niacincs.html).
Numerous reports indicate niacin might help prevent
atherosclerosis, diabetes and hypercholesterolemia. Niacin is effective in assisting burn-wound recovery, and in
the prevention of cataracts and skin cancer. Unfortunately, the molecular basis for most of these health
benefits remains unclear.
Niacin is a building block for the generation of cellular
NADC (nicotinamide adenine dinucleotide), which is well
known for its role as a carrier of two electron equivalents
for the oxidation of glucose and ultimate synthesis of ATP
during mitochondrial oxidative phosphorylation. In recent
years, NADC has emerged as a key player in signaltransduction pathways that do not involve the well-known
redox properties of the nicotinamide ring [1]. An interesting example is the Sir2 (silent information regulator 2) or
sirtuin family of NADC-dependent protein deacetylases.
Unlike other protein deacetylases, sirtuins require NADC,
and catalyze a reaction that cleaves nicotinamide from the
ribose ring and transfers the acetyl group from the N-3
lysine side chain to yield a unique metabolite O-acetylADPr (OAADPr) (Figures 1 and 2). Although the potential
spectrum of cellular functions for OAADPr remains to be
established, two reports hint at its biological importance.
Microinjection of OAADPr into starfish oocytes causes a
block in maturation, whereas microinjection into blastomeres yields a block in cell-cycle progression [2]. In a more
Corresponding author: Denu, J.M. (jmdenu@wisc.edu).
Available online 20 July 2005

recent study, OAADPr was shown to facilitate the

assembly of the yeast silencing complex, which consists
of silent-information-regulator proteins 2, 3 and 4 (Sir2,
Sir3 and Sir4) (Figure 1). A combination of genetic and
biochemical studies have demonstrated that yeast Sir2
(ySir2) silences genes within heterochromatin (unique
structural chromatin domains that repress transcription)
by functioning as a potent histone deacetylase [3]
(Figure 1). Deacetylation of heterochromatic regions is
essential for maintaining gene silencing. Sir3 and Sir4
display no homology to Sir2, but form a supramolecular
structure with Sir2 that spreads across the silent,
hypoacetylated loci. Interestingly, ySir2 and orthologs in
flies and worms also mediate lifespan extension [4].
Although the silencing effects in yeast are likely to be
caused to histone deacetylation by targeted ySir2
enzymes, the molecular basis for lifespan extension
remains elusive.
Histone
acetyltransferase
Acetyl-CoA
Ac

CoA
Ac

Gene
activation
Ac

Ac

Ac

Chromatin
Sir3

Sir3

Sir3

OAADPr

OAADPr

OAADPr

Sir2
Sir4

Sir2
Sir4

Sir2
Sir4

Gene
silencing

ySir2
deacetylation

OAADPr

NAD+

+
Nicotinamide
NAD+ salvage
pathway
Ti BS

Figure 1. Proposed function and regulation of yeast Sir2 in gene silencing. Histone
acetylation by histone acetyltransferases promotes gene activation. Sir2 is
recruited to silent chromatin; it deacetylates histones and forms a supramolecular
complex with Sir3 and Sir4 across silent chromatin to maintain a transcriptionally
repressive state. Deacetylation by Sir2 requires NADC and yields the additional
products nicotinamide and O-acetyl-ADP-ribose (OAADPr). Nicotinamide is a
potent inhibitor of the reaction, but can be recycled back to NADC via a NADCsalvage pathway. OAADPr binds to the Sir complex and might facilitate assembly of
the appropriate silencing structure.

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Sir2 proteins are found across all forms of life,

suggesting an ancient origin and a huge diversity in
functions among present-day sirtuins. The Sir2 homolog
from Salmonella deacetylates and activates the metabolic
enzyme acetyl-coenzyme A synthetase, thereby, enabling
this organism to survive on short-chain fatty acids [5]. The
archaeal chromatin protein Alba can be deacetylated by
the archaeal Sir2 enzyme, and a role in mediating
transcriptional repression has been proposed [6]. In
mammalian systems, the majority of investigations have
centered on the nuclear sirtuin SIRT1 (human nomenclature), one of seven known Sir2 homologs. Collectively,
these reports indicate that SIRT1 targets transcription
factors that are dynamically regulated by reversible
acetylation [4]. For example, the tumor-suppressor protein p53 is deacetylated by SIRT1, leading to an
attenuation of the p53-dependent apoptotic pathway
induced by cell stress [79]. SIRT1 negatively regulates
damage-responsive Forkhead transcription factors, promoting cell survival under stress [1012]. Also, SIRT1
controls the gluconeogenic and glycolytic pathways in
liver in response to fasting signals via the transcriptional
coactivator PGC-1a (peroxisome proliferator-activated
receptor-g coactivator-1a) [13,14]. Given the important
physiological processes that are regulated by sirtuins,
there has been considerable interest in understanding the
mechanisms of catalysis and regulation.
Inhibition of sirtuins by nicotinamide
New biochemical and structural studies have provided
enormous insight into catalysis of sirtuins and the
potential for regulation by various NADC metabolites.
Most notable with respect to the cellular regulation of
sirtuins is their dramatic inhibition by nicotinamide. First
analyzed in several kinetic studies [1517] and recently by
X-ray crystallographic analysis [18], nicotinamide and
several of its derivatives are powerful mechanism-based
inhibitors. The inhibition constants vary between homologs, but usually fall within the 20200-mM range, which
makes nicotinamide the most potent general inhibitor
described to date. Accordingly, nicotinamide has been used
to inhibit cellular sirtuins in a wide variety of experiments
in yeast and mammalian cells. For example, Bitterman
et al. [19] showed that nicotinamide-inhibited yeast
silencing increased rDNA recombination and shortened
replicative lifespan to that of a Sir2 mutant.
Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide
ribosyl bond, cleavage of an amide bond and transfer of the
acetyl group ultimately to the 2 0 -ribose hydroxyl of ADP
ribose (for reviews, see Refs [20,21]). Based on a series of
biochemical studies, an ADP-ribosyl-acetylpeptideenzyme intermediate (O-alkyl-amidate) is proposed to
form in the active site after initial cleavage of the NADC
nicotinamide ring (Figure 2). As the first product released
by the enzyme, nicotinamide functions as a classical noncompetitive product inhibitor and can inhibit the reaction
in the absence of the other two products, OAADPr and
deacetylated protein. The biochemical basis for inhibition
is the ability of nicotinamide to rebind and intercept the
O-alkyl-amidate intermediate before deacetylation, which
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Vol.30 No.9 September 2005

is, essentially, a simple reverse of the first half of the

reaction. In the process, nicotinamide reacts with the
intermediate (transglycosidation), thus driving the reaction
back towards the regeneration of NADC [16,17]. Although
a distinct allosteric site for nicotinamide inhibition has
been suggested [19,22], biochemical and new structural
evidence supports this model for nicotinamide inhibition
(Figure 2). However, confusion seems to arise from a
misunderstanding of the difference between a noncompetitive dead-end inhibitor and a non-competitive
product inhibitor [23]. Nicotinamide is a product inhibitor and, thus, can reverse the first half of the deacetylation
reaction. By contrast, a non-competitive dead-end inhibitor binds to both the free (Vmax/Km kinetic effect) and
substrate-bound forms (Vmax kinetic effect) of the enzyme,
but cannot proceed through any catalytic step (i.e. deadend complex is formed.) The non-competitive nature of
nicotinamide inhibition arises from nicotinamide binding
to, and reacting with, the intermediate form of the
enzyme, and results in the lowering of the amount of
enzyme capable of producing products (Vmax kinetic
effect). Because nicotinamide can react with the intermediate and increase the levels of the ternary complex at
the expense of the free enzyme, a kinetic effect on the
Vmax/Km constant is observed. The combined effects yield
non-competitive inhibition.
Although the first wave of sirtuin X-ray structures did
not reveal an intact nicotinamide moiety, new structural
data have provided direct evidence for the site of
nicotinamide binding [18,22,24] (Figure 3). Structures
from the Wolberger laboratory [18] show that free
nicotinamide binds in the conserved C pocket (Figure 3b),
which was previously shown to be important for NADC
binding and catalysis [22,2426]. Structural evidence
from both the Marmorstein and Wolberger laboratories
demonstrates that the nicotinamide moiety of NADC [24]
or an NADC analog (carba-NADC) [22] binds within the
C pocket (Figure 3a). Taking into account all available
structural and biochemical evidence, the nicotinamideriboside portion of NADC might adopt more than one
conformation depending on the presence of other ligands
or the solution conditions. When an acetyl-peptide binds,
conformational rearrangements in the enzyme promote
the productive binding of NADC and the nicotinamide is
forced into the buried C site, promoting cleavage of the
ribosyl-nicotinamide bond and protecting the resulting
reactive intermediate (O-alkyl amidate) from non-specific
hydrolysis (Figure 3a). Nicotinamide is released from the
enzyme, leading to the subsequent attack of the ribose
2 0 -OH on the intermediate and water addition to yield the
final products of the reaction (Figure 2). Zhao et al. [22]
proposed an alternative model for nicotinamide inhibition
whereby nicotinamide binds to, and reacts with, the
enzyme intermediate at a location distinct from the
C site. If, indeed, the C site is the location of nicotinamide
binding during the cleavage of the glycosidic bond, then
the law of micro-reversibility dictates that nicotinamide
must rebind and react from this C site, which argues
against this alternative model. The proposed catalytic
mechanism (Figure 2) supports a simple model for
nicotinamide inhibition of sirtuins that involves a single

Update

TRENDS in Biochemical Sciences

NAD+
H2N

481

NH2

NH2

O OO O
P
P
O
O
O

Vol.30 No.9 September 2005

N
+

Sir2
O

HO

OH

HO

HO

OH

OH
H3C

N
H

Enzyme ternary complex

H 3C

N
H
Acetylated substrate

H2N
H 2N
O

OH

O
N
Nicotinamide
release

Nicotinamide
attack

O
HO

O
CH3

H2O

2-O-acetyl-ADP-ribose

NH

H3C
HO
His

H2N

O
H

C1 O-alkylamidate
intermediate

Deacetylated product

Ti BS

Figure 2. Proposed mechanism for NADC-dependent protein deacetylation catalyzed by sirtuins. Biochemical studies support a two-step chemical mechanism involving the
initial formation of a 1 0 -O-alkylamidate adduct formed between the acetyl group and the nicotinamide ribose of NADC. Acetyl transfer to the 2 0 ribose and addition of water
yields deacetylated peptide and 2 0 -O-acetyl-ADP-ribose, a potential second messenger (pathway a). Nicotinamide is a potent product inhibitor of the reaction and can
regulate the cellular rate of deacetylation. Nicotinamide inhibits the deacetylation reaction by rebinding the O-alkylamidate intermediate form of the enzyme and attacking
the intermediate (transglycosidation) to regenerate starting reagents (pathway b).

nicotinamide-binding site, the same one involved in binding

the nicotaminde ring of co-substrate NADC. Although the
alternative model proposed by Zhao et al. [22] cannot be
ruled out, a second allosteric nicotinamide-binding site has
not been demonstrated, neither is such a binding site
required to explain the kinetic and biochemical data [16,17].
(a)

The specificity of the nicotinamide-binding pocket has

been investigated [1618,27]. Sirtuins demonstrate exquisite preference for the nicotinamide ring, whether for
potent inhibition as the free base or for co-enzyme use
during deacetylation. Various nicotinamide and pyridine
analogs exhibit sirtuin inhibition, but the potency is
(b)

Figure 3. Sir2 structures with bound ligands. (a) The structure of the yeast Sir2 homolog HST2 bound to the acetylated histone H4 peptide and a non-hydrolyzable NADC
analog, carba-NADC (PDB code: 1SZC) as reported by Zhao et al. [22]. (b) Structure of Thermotoga maritima Sir2 (Sir2Tm) bound to the acetylated p53 peptide and
nicotinamide (PDB code: 1YC5) as reported by Avalos et al. [18]. In both cases, the nicotinamide ring lies in the highly conserved C pocket [25] with the carboxamide moiety
hydrogen bonded to a conserved aspartic acid residue (Asp118 in HST2). Color code for secondary structural elements: green, a helices; gray, loops; yellow, b sheets. This
figure was generated using Swiss PDB Viewer v3.7 and POV-Ray v3.6.
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several orders of magnitude worse than nicotinamide. An

interesting example is the acid form of vitamin B3,
nicotinic acid, which displays almost negligible binding
to and inhibition of sirtuins. The physical basis for this
poor binding was revealed by the examination of the
nicotinamide-binding pocket, which harbors a negatively
charged aspartic acid residue that would create highly
unfavorable charge repulsion with nicotinic acid. With
nicotinamide, the aspartic acid interacts with the carboxamide portion of nicotinamide to promote tight binding
(Figure 3b). Avalos et al. [18] mutated this aspartic acid to
asparagine to explore the possibility of altering the coenzyme specificity [18]. Indeed, the Asp/Asn substitution created a mutant sirtuin capable of accepting
nicotinic acid adenine dinucleotide (NAAD) as a coenzyme. Compared with the native enzyme, this engineered enzyme is less sensitive to nicotinamide inhibition
but is more sensitive to inhibition by nicotinic acid.
Potent inhibition by nicotinamide involves two crucial
steps: (i) tight binding to the nicotinamide pocket and
(ii) the ability of bound nicotinamide to react with the
intermediate O-alkyl amidate [16]. Recently, Sauve et al.
[28] took advantage of this inhibitory mechanism by
identifying a nicotinamide analog, isonicotinamide, that
competes for free nicotinamide binding but does not react
appreciably with the enzyme intermediate [28]. The net
effect is a compound that can relieve nicotinamide
inhibition by preventing nicotinamide binding but still
enables significant progression through deacetylation.
Because isonicotinamide only weakly competes with
nicotinamide binding, high-mM concentrations of isonicotinamide are required to observe a release from nicotinamide-dependent inhibition in vitro and in yeast-silencing
assays [28]. Nonetheless, these results support the
rationale to develop nicotinamide derivatives that harbor
these properties but bind to sirtuins with higher affinity
and specificity.

Concluding remarks
The unique NADC-dependent sirtuin reaction and its
sensitivity to cellular nicotinamide levels provide an
exquisite mechanism for regulating the rate of deacetylation and the production of OAADPr. The recent studies
highlighted here have provided a molecular understanding of nicotinamide inhibition and its connection to the
catalytic mechanism of deacetylation. Together with
recent cellular studies, these data suggest new physiological roles for niacin and the possibility that a portion of
the health benefits of niacin feed through pathways
mediated by sirtuins. However, future studies are needed
to establish the physiological link between these health
benefits and sirtuin-regulated pathways. Perhaps most
telling will be to establish whether niacin treatment
directly inhibits sirtuins or, alternatively, up-regulates
these enzymes via increased NADC synthesis. It is
important to mention that, although only nicotinamide
inhibits sirtuins, both nicotinic acid and nicotinamide lead
to increased cellular NADC production. To help clarify
these issues, a complete understanding of NADC metabolism with quantification of relevant metabolites and
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Vol.30 No.9 September 2005

intermediates, and how these impinge on sirtuinmediated pathways will be essential.

Acknowledgements
I thank Brian Smith for generating Figure 2 and the anonymous referees
for their helpful comments on the manuscript. This work was supported
by National Institutes of Health grant GM65386.

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