NAD biosynthesis

A close look at NAD biosynthesis

A close look at NAD biosynthesis
Andrea Mattevi
Two new studies on the structure of an enzyme involved the synthesis of mammalian NAD shed new light on the
evolutionary and biochemical complexity of this fundamental metabolic pathway.
NAD is one of the ‘oldest’ molecules in the history
of biochemistry (Fig. 1a). Its discovery and
biochemical characterization go back to the first
half of the twentieth century. In the past several
years, there has been a sort of renaissance
in the biochemistry of NAD, with the discovery
that in addition to its well-established role in
redox biochemistry and energetic metabolism,
nicotinamide cofactors can function as signaling
molecules in a variety of cellular processes1.
NAD is the substrate in mono- and poly-ADP
ribosylation reactions that lead to the covalent
modification of proteins. Likewise, NAD is the
substrate of sirtuins, enzymes that catalyze the
deacetylation of histones and other proteins.
In addition, NAD is the precursor of signaling
molecules such as cyclic ADP-ribose. All
these reactions potentially lead to a progressive
depletion of the intracellular levels of NAD, and
there is therefore a continuous demand for NAD
biosynthesis to maintain NAD homeostasis2.
Interest in the pathways for NAD biosynthesis
has been further augmented by the discovery of
a link between the activity of the biosynthetic
enzymes and lifespan3. On pages 582 and 661 of
this issue, Kahn et al.4 and Wang et al.5 independently
report on the structural characterization
of mammalian nicotinamide phosphoribosyltransferase
(Nampt), a central enzyme in
NAD biosynthesis (Fig. 1b). Comparison with
the structures of other phosphoribosyltransferases6–
8 reveals various unexpected features
that help to explain the substrate specificity
of mammalian Nampt, which specifically acts
on nicotinamide and not on its derivatives or
precursors (Fig. 1). These features have implications
for understanding the molecular evolution
of the phosphoribosyltransferase class
of enzymes. Furthermore, the analysis of an
enzyme-inhibitor complex4 opens the way to
inhibitor-design studies.
Depending on the organism, three different
building blocks (quinolinic acid, nicotinic acid
and nicotinamide) can be used for the biosynthesis
of NAD (Fig. 1). Quinolinic acid is the
precursor for the so-called de novo pathway; in
eukaryotes, it is normally synthesized starting
from tryptophan, whereas in bacteria it is synthesized
from L-aspartate and dihydroxyacetonephosphate.
In the so-called salvage pathways,
NAD is synthesized starting from nicotinic acid
and nicotinamide that are produced by the various
reactions that degrade and consume NAD
(Fig. 1). Quinolinic acid, nicotinic acid and
nicotinamide are used by phosphoribosyltransferases
to produce nicotinic acid mononucleotide
or nicotinamide mononucleotide6–8. These
molecules are then converted to the corresponding
dinucleotides through adenylation reactions
catalyzed by mononucleotide adenylyltransfer-
The author is in the Department of Genetics and
Microbiology, University of Pavia, Via Ferrata 1,
27100 Italy.
Figure 1 NAD and its biosynthesis (a) Chemical formula of NAD and its derivatives. (b) Simplified
schematic view of the reactions involved in NAD biosynthesis, with emphasis on the reactions
described in the text.
Katie Ris
© 2006 Nature Publishing Group
ases9–12. Finally, NAD synthase catalyzes the amidation
of nicotinic acid adenine dinucleotide to
generate the NAD molecule. Recent years have
witnessed tremendous advances in the structural
biology of NAD biosynthesis, to the point that
the three-dimensional structures of most of the
enzymes involved in the pathway are known13.
One of the most fascinating results revealed by
these structural and biochemical investigations
concerns the diversity in the specificities of the
enzyme reactions. There are specific phosphoribosyltransferases
for each of the precursors (Fig.
1b), the mammalian Nampt described by Kahn
et al.4 and Wang et al.5 being strictly specific for
nicotinamide. Likewise, adenylyl transferases
differ in specificity depending on the organism;
the Escherichia coli enzyme preferentially acts
on nicotinic acid mononucleotide, whereas the
mammalian and archaeal proteins are less stringent,
acting on both nicotinamide and nicotinic
acid mononucleotides9–12.
Enzymes of the phosphoribosyltransferase
class catalyze the addition of the phosphoribosyl
moiety derived from phosphoribosylpyrophosphate
to their specific substrates. The structures
of bacterial quinolinic acid and nicotinic acid
phosphoribosyltransferases have been described
before6–8, and with the structures reported in
this issue of the mammalian Nampt4,5, it has
become possible to compare the phosphorib
osyltransferases involved in bacterial and/or
mammalian NAD biosynthesis. Although the
three enzymes share a similar overall folding
topology and quaternary structure, they differ
substantially in the details of their architecture
and active site geometry, implying that, despite
their similar enzymatic activities, they have
diverged considerably in the course of evolution.
Of particular interest is the comparison of
the active sites. Nampt is characterized by the
presence of an aspartate residue that interacts
with the nitrogen atom of the carbamide group
of the nicotinamide substrate. In nicotinic acid
phosphoribosyltransferase, this aspartate is
replaced by a serine, suggesting that it is one of
the crucial elements that dictates the specificity
for either nicotinamide or nicotinic acid in
these enzymes (Fig. 1b). Indeed, as shown by
Khan et al.4, the Asp→Ser mutation confers a
dual specificity to the mutated Nampt, which is
able to act on both nicotinamide and nicotinic
acid. In this regard, it will be of interest to look
at the structure of the mutant enzyme in complex
with nicotinic acid, to see how the mutation
enables the protein to bind in a catalytically
competent orientation.
However, the story is even more complicated.
A 10-residue insertion in an active site loop of
Nampt induces a shift of a β-strand that modifies
the shape of the active site cleft. This structural
feature is another element that contributes to
the specificity of mammalian Nampt. Thus,
even a simple variation in substrate specificity
such as the discrimination between a carbamide
and a carboxylate substituent can be associated
with rather elaborate changes in the conformation
of the substrate- recognition site. This
notion is further illustrated by the various adenylyltransferase
enzymes involved in the NAD
biosynthetic pathways9–12. Comparison between
the bacterial nicotinic acid adenylyl transferase
and the mammalian nicotinamide/nicotinic
acid adenylyl transferase reveals changes in both
the overall structure and the conformation of the
active site residues. Thus, for the phosphoribosyltransferases
as well as the adenylyl transferases,
differences in substrate specificity seem to result
from the additive effects of subtle alterations
in the active site conformation and geometry,
including the presence of ordered waters that
mediate the interactions between the substrate
and the protein.
NAD biosynthesis is emerging as one of the
most thoroughly investigated metabolic pathways
from both a biochemical and a structural
standpoint1,13. Its characteristic feature is that
it does not consist of a single defined pathway
conserved in all organisms. The ‘logic’ and
sequence of reactions are generally conserved,
but depending on the organism, NAD biosynthesis
starts from different precursors and
takes different routes for the recycling of the
products of NAD breakdown. Consequently,
the NAD biosynthetic enzymes have acquired
different specificities in the different organisms.
The main theme emerging from comparative
analysis of the structures of these enzymes
is that of unexpected complexity. Changes
in substrate specificity underlie a substantial
degree of evolutionary divergence that results
in large changes in the overall protein structure
as well as in the conformation and geometry of
the active site. These features should make the
NAD biosynthetic enzymes ideal systems for
in vitro evolution studies.
1. Berger, F., Ramirez-Hernandez, M.H. & Ziegler, M.
Trends Biochem. Sci. 29, 111–118 (2004).
2. Magni, G. et al. Cell. Mol. Life Sci. 61, 19–34 (2004).
3. Anderson, R.M. et al. Science 302, 2124–2112
4. Khan, J.A., Tao, X. & Tong, L. Nat. Struct. Mol. Biol. 13,
582–588 (2006).
5. Wang, T. et al. Nat. Struct. Mol. Biol. 13, 661–662
6. Sharma, V., Grubmeyer, C. & Sacchettini, J.C. Structure
6, 1587–1599 (1998).
7. Shin, D.H. et al. J. Biol. Chem. 280, 18326–18335
8. Chappie, J.S. et al. Structure 13, 1385–1396 (2005).
9. Zhang, H. et al. Structure 10, 69–79 (2002).
10. Zhou, T. et al. J. Biol. Chem. 277, 13148–13154
11. Saridakis, V. et al. J. Biol. Chem. 276, 7225–7232
12. Garavaglia, S. et al. J. Biol. Chem. 277, 8524–8530
13. Rizzi, M. & Schindelin, H. Curr. Opin. Struct. Biol. 12,
709–720 (2002).
The author is in the Department of
Biochemistry, Division of Nucleic Acids
Enzymology, Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854, USA.
The Pol II initiation complex: finding a place to start
Michael Hampsey
Yeast RNA polymerase II has been proposed to ‘scan’ template DNA for transcription start sites. A new study
mapping promoter DNA trajectory through the preinitiation complex suggests a mechanism for how this occurs.
Over the past six years, a series of papers
have provided high-resolution structures of
yeast RNA polymerase II (Pol II) alone1–4
and in elongation complexes with DNA and
RNA5,6. Recently, a 2.3-Å-resolution image
of a transcribing Pol II complex was solved,
offering remarkable insight into transcription
mechanism7. Missing from the picture, however, is the trajectory of DNA through the Pol II preinitiation complex (PIC), leaving us an incomplete structural image of transcription initiation. On page 603 of this issue, Miller and Hahn address this by mapping
the path of promoter DNA through a functional PIC using DNA-tethered cleavage
probes8. Their results suggest a mechanismfor how yeast Pol II scans DNA for transcription start sites.
© 2006 Nature Publishing Group

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