From the Department of Biochemistry and ¶ Howard
Hughes Medical Institute, University of Texas Southwestern Medical
Center, Dallas, Texas 75390 and § Integrated Genomics, Inc.,
Chicago, Illinois 60612
Received for publication, January 3, 2003, and in revised form, February 4, 2003
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ABSTRACT |
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Pyridine dinucleotides (NAD and NADP) are
ubiquitous cofactors involved in hundreds of redox reactions essential
for the energy transduction and metabolism in all living cells. In
addition, NAD also serves as a substrate for ADP-ribosylation of a
number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing
regulation, and for cyclic ADP ribose (cADPR)-dependent
Ca2+ signaling. Pyridine nucleotide
adenylyltransferase (PNAT) is an indispensable central enzyme in the
NAD biosynthesis pathways catalyzing the condensation of pyridine
mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or
NaAD). Here we report the identification and structural
characterization of a novel human PNAT (hsPNAT-3) that is located in
the cytoplasm and mitochondria. Its subcellular localization and tissue
distribution are distinct from the previously identified human nuclear
PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the
catalytic mechanism of the enzyme. The characterization of the
cytosolic human PNAT-3 provided compelling evidence that the final
steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and
mitochondria, potentially contributing to their NAD/NADP pool.
The coenzymes
NAD+(H)1 and
NADP+(H) have been known for many decades as the major
hydrogen donor or acceptor in hundreds of metabolic redox reactions
throughout the cell. Together these nucleotides have a direct impact on
virtually every cellular metabolic pathway. Additionally, NAD serves as
a substrate for the covalent modification of nuclear proteins by
ADP-ribosylation, a process involved in DNA repair and the regulation
of genomic instability (1-3). Recently, many new exciting functions
have been discovered for this long-known molecule. These include its
role as co-substrate in Sir2-mediated histone deacetylation involved in
gene silencing regulation and in increasing the lifespan of species
ranging from yeast, to worm, to certain mammals (4, 5). Moreover,
several derivatives of NAD and NADP were found to be potent
intracellular calcium-mobilizing agents and are involved in a variety
of Ca2+-signaling pathways (6-8). These recent
developments brought a significant amount of additional interest to the
investigation of cellular NAD biosynthesis and regulation.
NMN and/or NaMN adenylyltransferase (NMNAT and/or NaMNAT, collectively
named pyridine nucleotide adenylyltransferase, or PNAT) is an
indispensable enzyme catalyzing the central step of all NAD
biosynthesis pathways (9, 10). It links the AMP moiety of ATP with the
nicotinamide mononucleotide (NMN, or its deamidated form NaMN) to form
the dinucleotide product NAD (or deamido-NAD, NaAD). A practical aspect
of human PNAT function is that it catalyzes the rate-limiting step in
the metabolic conversion of the anticancer agent tiazofurin to its
active form TAD (tiazofurin adenine dinucleotide, an NAD analog) (11).
The development of tiazofurin resistance has been shown to relate
mainly to a decrease in PNAT activity (12). Recently, it has also been
found that human nuclear PNAT (hsPNAT-1, or NMNAT-1 as in Raffaelli
et al. (25)) may play an important role in delaying
the Wallerian degeneration in injured axons and synapses: the causative
mutation in the slow Wallerian degeneration mouse is found to be a
chimeric gene Ube4b/NMNAT encoding hsPNAT-1 fused with the
N-terminal region of the ubiquitination factor E4B (13, 14).
Significant progress has been made during the last few years in the
enzymological and structural studies of PNATs in various organisms
across all three kingdoms of life (15-22). In human, two PNAT isoforms
(gi 11245478 and gi 12620200) have been cloned and purified, and
their kinetic properties were analyzed (23-26). Human nuclear PNAT-1
was shown to present exclusively in the nuclei and express at high
levels in heart, skeletal muscle and, to a lesser extent, in kidney and
liver (23). Human PNAT-1 has also been reported to interact
specifically with poly(ADP-ribose) polymerase (24, 27) and may be
subjected to further regulation by phosphorylation (24). Crystal
structures of hsPNAT-1 have been solved in its apo form (20) and in
complex with NMN (21), NAD, NaAD, or TAD (19). These structures
revealed a hexameric organization of the protein and provided a
structural basis for its dual specificity in recognizing both NMN and
NaMN substrates (19). Another isoform of human PNAT, hsPNAT-2 (or
NMNAT-2 as in Raffaelli et al. (25)) is distinct from the
nuclear PNAT-1 (25, 26). It contains 307 residues (versus
279 in hsPNAT-1) and shares 34% sequence identity with the nuclear
enzyme. Its tissue expression level is generally low, and it is thought
to be primarily expressed in the nervous systems (25). The enzymatic
activity of hsPNAT-2 activity is much lower compared with the nuclear
form. Both human PNAT isoforms recognize NMN and NaMN substrates
equally well; therefore, they are capable of participating in the
de novo and salvage/recycling routes of NAD biosynthesis via
either NMN or NaMN intermediates.
Historically, PNAT activity has been detected in various animal
tissues, including mammary gland, testis, liver, placenta, kidney,
thymus, erythrocyte, lymphocyte, and retina (28-33). Although the
localization of PNAT activity has been asserted in nuclei (34, 35), a
substantial cellular NAD pool is stored in mitochondria (36, 37), and
PNAT activity in rat liver mitochondria has also been reported (38).
Here we report the identification and characterization of a novel human
PNAT, named hsPNAT-3, which is located in the cytosol and mitochondria.
Structural analysis of hsPNAT-3 and its complexes with substrates and
products provided critical insight in the catalytic mechanism of the
adenylyltransfer reaction, which are likely to be shared among all
PNATs and some other (T/H)IGH motif-containing adenylyltransferases.
Materials--
The poly(A)+-RNA filters of 10 human
tissues and 10 human cancer cell lines, hybridization buffer, and
washing buffer were purchased from Ambion (Austin, TX). The
Pfu DNA polymerase was from Stratagene (La Jolla, CA). The
Rediprime II DNA labeling kit was from Amersham Biosciences (UK). The
pPROEX-1a prokaryotic expression vector was from Invitrogen (Carlsbad,
CA). Nickel-nitrilotriacetic acid-agarose was from Qiagen (Valencia,
CA). The pEGFP-N1 mammalian expression vector was from
Clontech (Palo Alto, CA). The MitoTraker® Red
mitochondria-specific fluorescent dye was purchased from Molecular Probes (Eugene, OR). The TEV protease used to remove the His tag of the
expressed recombinant proteins was purified in our laboratory from an
expression vector kindly provided by Dr. Dave Waugh (NCI, National
Institutes of Health, Frederick, MD). The Wizard I and II
crystallization screening kits were from Emerald Biostructures (Bainbridge Island, WA).
Expression and Purification of HsPNAT-3--
The hsPNAT-3
cDNA was amplified from a human brain cDNA library
(Clontech) and inserted into the pPROEX prokaryotic
expression vector (Invitrogen). The resultant pPROEX-hsPNAT3 vector was
then transformed into Escherichia coli DH10 NMNAT- and NaMNAT-coupled Assay--
The hsPNAT-3 activity with
both substrates NMN and NaMN was analyzed using a continuous
assay coupling NAD formation to alcohol dehydrogenase-catalyzed
conversion of NAD to NADH as previously described (39, 40). Briefly,
the assay was performed in UV-transparent plastic cuvettes in the
six-cuvette autosampler of a DU-640 spectrophotometer (Beckman,
Fullerton, CA) at 37 °C. The 500-µl reaction contained 100 mM HEPES-KOH, pH 7.5, 115 mM ethanol, 40 mM semicarbazide, 2 mM ATP, 3 units of alcohol
dehydrogenase (Sigma), and ~0.01 µg of hsPNAT-3. The reaction was
started by adding 10 µl of 20 mM NMN and monitored at 340 nm over a 20-min period. The NADH extinction coefficient of 6.22 mM Northern Hybridization--
The DNA template of hsPNAT-3 used
for synthesizing a hybridization probe was amplified by PCR from the
pPROEX-hsPNAT3 plasmid. About 25 ng of DNA was used for probe synthesis
in the presence of [32P]CTP using the Rediprime II system
(Amersham Biosciences). The synthesized probe was purified with a Micro
Bio-spin 30 column (Bio-Rad) to remove the non-incorporated
nucleotides. The two poly(A)+-RNA filters (the normal cell
multiple tissue blot and multiple cancer cell line blot from Ambion)
were prehybridized at 42 °C for 30 min before being hybridized with
the 32P-labeled probe at 42 °C for 16 h. After
hybridization, the filters were washed according to the manufacturer's
instruction. The autoradiography was set at Mammalian Cell Culture and GFP Localization--
HEK293 (human
kidney epithelial) and HeLa cells (kind gift from Dr. Helen Yin, UT
Southwestern Medical Center at Dallas, TX) were cultured in the
Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal
bovine serum (Harlan Bioproducts for Science). The cells were incubated
at 37 °C with 5% CO2. To make the C-terminal GFP fusion
mammalian expression constructs for hsPNAT-1, hsPNAT-2, and hsPNAT-3,
the cDNA of each of the three genes was amplified by PCR from their
respective prokaryotic expression plasmids and inserted into the
pEGFP-N1 mammalian expression vector (Clontech). The resultant vectors pEGFP-hsPNAT1, pEGFP-hsPNAT2, and pEGFP-hsPNAT3 were transfected into cells using SuperFect reagent (Qiagen) when the
cell density reached about 70% confluence. The localization of the
expressed GFP fusion proteins was examined after 2-day transfection
with a Zeiss Axiovert 35 fluorescence microscope. The co-localization
of hsPNAT-3 and mitochondria-specific dye was observed under a Zeiss
LSM 510 confocal fluorescence microscope.
Crystallization and Data Collection--
The hsPNAT-3 crystals
were obtained using the hanging drop, vapor diffusion method. 1.5 µl
of hsPNAT-3 (20 mg/ml) was mixed with 1.5 µl of reservoir solution
containing 100 mM sodium cacodylate, pH 6.0-7.0, 200 mM Li2SO4, and 20-25%
polyethylene glycol 400, and equilibrated against the reservoir. The
crystals of the complexes of hsPNAT-3 with various substrates and
products, including NMN, AMP-CPP, NAD, NaAD, and a ternary
complex with both NMN and AMP-CPP, were obtained by co-crystallization.
All crystals were directly frozen in liquid propane, and the
diffraction data were collected on a RAXIS-IV image plate detector
equipped with a Rigaku-H3R x-ray generator and Osmic mirrors at a
temperature of 100 K. The data were processed with the HKL2000 package
(41), and the space group was later determined to be
P43212 with cell dimensions a = b = 79.36 Å, and c = 146.2 Å. There are two hsPNAT-3 monomers in the
asymmetric unit. The crystal data of all six crystals are listed in
Table I.
Structure Determination and Refinement--
The structure of the
apo hsPNAT-3 was determined by the molecular replacement method in CNS
(42) using the coordinates of the nuclear hsPNAT-1 monomer as the
initial search model. The cross-rotation search found two solutions
with correlation coefficients (c.c.) of 0.0686 and 0.0556, respectively, which were significantly higher than the rest of the
solutions (the next highest peak had a c.c. of 0.0325). After
translation search and rigid body refinement with the two monomers
located, the c.c. was 0.647 and the R-factor was 0.39. Subsequent refinement and model building were then carried out using
the programs CNS (42) and O (43). The refined apo-hsPNAT-3 structure
was used as the initial model for the refinement of the complex
structures. Various ligands and solvent molecules were included in the
models according to the difference densities at later stages of the
refinement. The refinement statistics of all six structures are listed
in Table I.
A BLAST search (44) of the non-redundant protein sequence data
base (NCBI, National Institutes of Health, Bethesda, MD) using hsPNAT-1
(gi 11245478) as query found several human homologous proteins that
also possesses the signature (T/H)IGH and ISSTXXR adenylyltransferase motifs (18, 45), suggesting that these proteins may
also encode for PNAT (see Supplemental Material). In particular, a
252-amino acid human protein sequence (gi 14029540), termed here
hsPNAT-3, shares 50% identity with hsPNAT-1 and 82% identity with a
mouse protein (gi 13543122) that presumably is the mouse ortholog of
hsPNAT-3. We have cloned and overexpressed hsPNAT-3 and experimentally
confirmed that indeed it has the predicted adenylyltransferase
activity. Like the two other isoforms, hsPNAT-3 has comparable activity
with both physiological substrates (5.6 units/mg for NaMN and 4.7 units/mg for NMN at saturating concentration of either substrate). Both
activities of hsPNAT-3 are lower compared with hsPNAT-1 (38 and 30 units/mg, respectively), but being ~10-fold higher compared with
hsPNAT-2. Together with the previously reported hsPNAT-1 and hsPNAT-2,
three human PNAT isoenzymes are now identified. To understand the
cellular and physiological roles of these different human PNATs, we
studied the tissue distributions and subcellular localization of the
three different hsPNATs by Northern hybridization and expression of GFP
fusion proteins in cultured kidney epithelial HEK293 and HeLa cells.
Northern Blot Analysis of hsPNAT-3--
The overall mRNA
levels of hsPNAT-3 in normal tissues and cancer cell lines were low
compared with hsPNAT-1 (23) (Fig. 1). Clear hybridization bands of a 1.1-kb transcript were detected in
mRNA blots of human lung and spleen, and weaker reaction bands were
detected in placenta and kidney. There was almost no hybridization signal detected in all the cancer cell lines tested (data not shown).
This tissue distribution pattern is different from both human PNAT-1
and PNAT-2 (23, 25). There appears to be no overlapping in tissue
distributions between the three hsPNAT isoforms, suggesting that
expression of these isoenzymes are differentially controlled at
transcription level and each may have a distinct cellular and physiological role.
Subcellular Localization of the Three Human PNAT
Isoenzymes--
We monitored subcellular localization of the three
human PNAT isoenzymes tagged with GFP and overexpressed in HEK293 and
HeLa cells (Fig. 2). We found that
GFP-hsPNAT1 is located exclusively in the nuclei (data not shown),
consistent with the results from earlier immunofluorescent studies
(24). GFP-hsPNAT3 is shown to exist outside nucleus. Further
examination of the co-localization of GFP-hsPNAT3 with the red
mitochondria-specific dye showed that hsPNAT-3 is also present in
mitochondria (Fig. 2A). GFP-hsPNAT2, on the other hand, is
found to have a very specific localization outside nucleus that does
not overlap with the mitochondria-specific dye (Fig.
2B). Its exact location in the cell remains to be
determined.
The results obtained here demonstrate for the first time that two human
PNAT isoenzymes are expressed outside cell nucleus. Whereas the highly
compartmentalized localization of hsPNAT-2 remains to be determined,
hsPNAT-3 is shown to express in the cytosol and mitochondria. Together
with previous observations that a substantial cellular NAD pool is
stored in mitochondria (36, 37) and that PNAT activity has previously
been detected in rat liver mitochondria (38), the identification of a
cytosolic and mitochondria human PNAT-3 gives further evidence that at
least part of the NAD biosynthesis pathways exists in human cytosol and mitochondria.
The Tetrameric Organization of hsPNAT-3--
To learn the
structural mechanisms of the function of hsPNAT-3, we have determined
three-dimensional structures of hsPNAT-3 in its apo form and in
complexes with each substrate (ATP analog AMP-CPP or NMN), product (NAD
or NaAD), and with both NMN and AMP-CPP. The overall structure of the
hsPNAT-3 monomer is very similar to the nuclear hsPNAT-1, with root
mean square deviation (r.m.s.d.) between the superimposed
C
The active site residue arrangement in hsPNAT-3 is also very similar to
that in hsPNAT-1. However, the higher order oligomerization states of
the two hsPNAT isoforms are different. There are two independent
hsPNAT-3 monomers in the asymmetric unit of the crystal. Inspection of
the crystal packing indicated that hsPNAT-3 forms a tetramer (Fig.
3B), which is consistent with the deduced molecular weight
from gel filtration chromatography. The hsPNAT-3 tetramer is tightly
packed, and the four monomers in the tetramer are related by a
D2 symmetry. One of the dimer interfaces (e.g.
between the yellow and orange monomers in Fig.
3B) is similar to that observed in hsPNAT-1 (19-21). A
similar dimer interface is also conserved in Bacillus
subtilis NaMNAT, which exists as dimers in solution (18). Although
this commonly shared dimerization interface buries a
1223-Å2 surface area on each monomer, a second type of
dimer interface (e.g. between the yellow and
cyan monomers in Fig. 3B) that is unique for
hsPNAT-3 buries about the same amount of surface area (1244 Å2). Both types of interface are largely polar in nature.
Similar to hsPNAT-1, hsPNAT-3 is also highly positively charged with a theoretical pI of 9.28, but these charges are rather dispersed and no
surface charge clustering is observed.
Ligand Binding-induced Conformational Changes--
Compared with
the apo-hsPNAT-3, all the substrate/product-bound hsPNAT-3 complexes
display some common trends in the ligand binding-induced conformational
changes (Fig. 4). The r.m.s.d. values
between C
The ligand binding-induced conformational changes near the NMN binding
site are local and mostly in the residues that make contact with NMN
(Fig. 4). Upon binding NMN or NAD/NaAD, residues 50-56 and 144-149
around the NMN binding site made various degrees of adjustment (usually
less than 1 Å) and in general closed in on the bound substrate so that
the contacts with the ligand are optimized. Similar ligand
binding-induced movements around the NMN binding site were also
observed in hsPNAT-1 (19, 20).
At the adenine binding site, however, local side-chain movements as
well as large main-chain conformational changes in loop 193-199
occurred upon binding the adenine containing ligands (Fig. 4). In the
apo-hsPNAT-1 structure, side chains of Met-21, Met-25, and
Arg-167 are pointed toward the adenine binding site and would have clashed with a bound adenylate ligand (Fig. 4). Val-196 located on
the loop 193-199 is also close to the adenine binding site. Upon
binding the ATP substrate or NAD/NaAD, the Arg-167 side chain is now
stacked against adenine ring, and the side chains of the two
methionines, Met-21 and Met-25, are rotated away. The loop 193-199 underwent large conformational changes with the Val-196 side
chain now moved outward by ~6 Å (Fig. 4). The new conformation of
loop 193-199 enables a hydrogen bond to be formed between N6 of
adenine and the main chain carbonyl of Asn-198 (see below).
It needs to be pointed out that such adenine nucleotide binding-induced
conformational changes in loop 193-199 were not observed in the
monomer B in the asymmetric unit. The conformation of this loop in
monomer B in the apo structure is similar to that of the ligand-bound
form, and no drastic conformational change occurs upon adenylate
binding. The reason for this asymmetry is not clear, but may be related
to the different crystal packing environments of the two independent
hsPNAT-3 monomers. The relatively small ligand binding-induced
conformational changes in both hsPNAT-1 and hsPNAT-3 are in contrast to
the drastic conformational changes observed in the bacterial (E. coli and B. subtilis) NaMNATs, which occur at both ATP
and NMN binding sites (17, 18).
The ATP Binding Site--
The bound AMP-CPP (a non-hydrolyzable
ATP analog) in the binary and ternary complexes adopts essentially
identical conformations and makes the same interactions with the
enzyme. The conformation of the AMP portion of AMP-CPP is also very
similar to that of the AMP moiety of the bound NAD. The detailed
interactions between bound AMP-CPP and surrounding protein atoms are
shown in Fig. 5A. In
particular, the ATP triphosphate tail makes extensive interactions with
the enzyme. The
In the hsPNAT-3 complexes with NAD or NMN, a sulfate molecule was
located near the bound ligand. Superposition with the ATP complex shows
that this sulfate molecule occupies a position near the The NMN Binding Site--
In the hsPNAT-3 and NMN binary complex,
alternative conformations exist in the phosphate portion of NMN in
monomer B (Fig. 5B). In one of the two conformers, the
phosphate is bent toward the pyridine ring, whereas in the second
conformer the phosphate is in a more extended conformation. The
distance between the phosphor atoms in the two conformers is 2.7 Å.
The NMN bound to monomer A adopts exclusively the "bent"
conformation. The reason for this asymmetric binding of NMN in the two
hsPNAT-3 monomers of the asymmetric unit is not clear. In monomer A
where NMN adopts the bent conformation, the side chain of Tyr-53
rotates about 20° compared with that in the monomer B to avoid
potential close contact with the NMN phosphate. As a result, the
side-chain hydroxyl of Tyr-53 moves 1.2 Å and is now hydrogen-bonded
to the NMN phosphate oxygen (2.53 Å) (Fig. 5C). Otherwise,
there are few interactions between protein atoms and NMN phosphate in
the bent conformation. In the extended conformation, the NMN phosphate
interacts with the main-chain amide and side-chain hydroxyl of Ser-14.
It also interacts with the Phe-15 main-chain amide through a water
molecule (Fig. 5C). Such extended conformation is also
observed in the NMN and NaMN binary complexes of
hsPNAT-1,2 and in the closely
related PPAT binary complex with phosphopantetheine (46). It appears
that the extended NMN conformation forms more interactions with the
protein and may be energetically more favorable. It was thus somewhat
unexpected that the NMN in the hsPNAT3·NMN binary complex adopted the
bent conformation in the absence of ATP. Notably, a sulfate molecule is
present in the NMN binary complex that partially overlaps with the
binding site of the ATP
The conformation of the nicotinamide ribose portion of the bound NMN is
essentially the same as the corresponding region in NAD. The detailed
interactions between NMN and protein is similar to that in hsPNAT-1 and
is shown in Fig. 5C. In particular, the pyridine ring stacks
against the Trp-148 side chain and forms a face-to-edge interaction
with Trp-90. The exocyclic carboxyamide group interacts specifically
with the main-chain amide of Thr-93 and main-chain carbonyl of Leu-147.
The Leu-147 side chain also forms van der Waals contacts with the
pyridine ring. Two highly conserved active site water molecules (w1 and
w2) that are seen in hsPNAT-1 and bacterial NMNATs are also conserved
in hsPNAT-3 and interact with the carboxyamide moiety of nicotinamide.
One of these water molecules (w1) is linked to the amide group of the
nicotinamide, main-chain amide of Lys-149, and the side-chain N Structural Basis for the Dual Substrate Specificity of
hsPNAT-3--
To understand the structural basis for the dual
substrate specificity of hsPNAT-3, we have solved the structures of
hsPNAT-3 complexed with either NAD or NaAD. As mentioned before, the
pyridine ribose portion of the dinucleotide occupies the same binding
site as that in the NMN binary complex. The interactions between the nicotinamide moiety and surrounding protein atoms as well as the two
active site water molecules are very similar to that observed in the
hsPNAT-1 (19) (Fig. 5C). Previously, we have found that in
hsPNAT-1·NaAD complex, besides the subtle local conformational changes, an active site water molecule also moved toward the
carboxylate, partially compensating the negative charge of NaAD (19).
In the hsPNAT3·NaAD complex, however, no active site water movement is observed that could provide additional interactions with the negatively charged carboxylate. Upon close inspection of the
surrounding protein groups, we found that one of the active site water
molecules (w1) that interacts with the carboxylate of NaAD is
hydrogen-bonded to His-152 (Fig. 5C). We hypothesize that
this water molecule may interact with the amide group of NAD as a
hydrogen acceptor and change to a hydrogen donor when interacting with
the carboxylate oxygen of NaAD, partially neutralizing the negative
charge of NaAD. Coordination to a histidine side chain should enable
this water molecule to perform this dual functional role.
The NMN·AMP-CPP Ternary Complex Structure and the Mechanism of
Adenylyltransfer Reaction--
In the hsPNAT-3 ternary substrate
complex structure, the conformation of the NMN molecule adopts the bent
conformation similar to that in the NMN binary complex. Steric
hindrance due to the presence of the ATP triphosphate tail appears to
prevent NMN from adopting an extended conformation when both substrates
are bound (Fig. 6A). The
AMP-CPP conformation is essentially the same as that of the AMP-CPP in
the binary complex. Compared with the respective binary complexes, the
B-factors are much higher for both substrates in the ternary
complex (Table I), indicating that
simultaneously bound substrates in the enzyme active site are less
stable and may be of partial occupancies. Additionally, the average
B-factor of protein atoms in the ternary complex is also
somewhat higher than that of the binary complexes, suggesting that the
enzyme might become more flexible upon binding of both substrates.
Notably, in the NMN and NaMN binary complexes obtained for
hsPNAT-12 and in the binary complex of the related E. coli PPAT with substrate phosphopantetheine (46), the
phosphorylated adenylyl acceptors all adopt the extended conformation
that overlaps the corresponding portion of the product. However, in the
hsPNAT-3 ternary substrate complex, the presence of the ATP
triphosphate tail makes the extended NMN phosphate conformation
impossible, and as a result the NMN phosphate has to adopt a highly
bent conformation. Because there are few interactions with the enzyme
that would stabilize the NMN phosphate in its bent conformation, we
assume that this conformation is less favorable or less stable than the
"extended" conformation. The alternative bent and extended NMN
conformations in the PNAT active site provided critical insight into
the catalytic mechanism of PNAT, which has been proposed to use an
in-line nucleophilic attack of the NMN phosphate on the ATP
The structure of hsPNAT-3 complexed with both AMP-CPP and NMN described
here is the first ternary substrate complex structure reported for any
(T/H)IGH motif-containing adenylyltransferase. The reaction
mechanism proposed here is likely to be shared among other PNATs and
some adenylyltransferases as well. It should be noted that the NMN
molecule in the hsPNAT3·NMN binary complex can also adopt the bent
conformation, indicating that the energetic difference between the two
conformations (bent versus extended) is marginal. It is
likely that several factors, including the tendency of NMN to adopt an
extended conformation, Mg2+ coordination, and interactions
with the surrounding protein atoms, all contribute synergistically to
facilitate the adenylyltransfer reaction.
Three Distinct hsPNAT Isoforms and NAD Metabolism in
Human--
The PNAT activity has been detected in a variety of animal
tissues, including mammary gland, testis, liver, placenta, thymus, kidney, retina, lymphocyte, and erythrocyte (28-33). Many reports have
shown that this activity resides in the nuclei (34, 35). However,
substantial cellular NAD pool is stored in mitochondria (36, 37, 48),
and PNAT activity has been reported from the rat liver mitochondria as
well (38). The first human PNAT characterized at the molecular and
atomic structural level contains a typical monopartate nuclear
localization signal sequence (NLS) and was shown to exist exclusively
in nuclei by the immunofluorescent studies (24) and by the localization
of the expressed GFP fusion protein (this study). This human nuclear
PNAT (hsPNAT-1) is highly expressed in heart and skeletal muscles and
weakly expressed in kidney, pancreas, and liver (23). The second human
PNAT isoform, hsPNAT-2, does not possess a typical NLS and has been
predicted to be cytosolic (25). Here we have shown that its
localization outside nuclei is highly compartmentalized and that it is
not expressed in mitochondria. The mRNA level of hsPNAT-2 is low
compared with the nuclear hsPNAT-1 and exists mostly in the nervous
system (25). The third human hsPNAT-3 reported here exists in cytosol and mitochondria, but not nucleus. Its tissue distribution is distinct
from hsPNAT-1 and hsPNAT-2 and is present in lung and spleen and, to a
lesser extent, in placenta and kidney, where no expression of the other
two hsPNAT isoforms were detected. All three hsPNAT isoenzymes are
expressed in cancer cells at much lower levels, which are assumed to be
close to the critical level for cell survival (29, 49). Consequently,
PNAT has been regarded as a potential target for anticancer
chemotherapy, because the minimal inhibitory concentration lethal to
cancer cells is not likely to cause serious damage to normal cells
(49).
Human PNAT-1 and -3 have close orthologs in mouse and probably in other
mammals as well (see Supplemental Material). They share 82% sequence
identity to their respective mouse orthologs. So far no mouse orthologs
has been found for hsPNAT-2, which may be due to the fact that the
sequencing of mouse genome is yet to be completed.
Currently most of the enzymatic steps involved in human NAD/NADP
biosynthesis are understood, and several relevant pathways have been
asserted. Genes encoding all of the steps in the five-stepped de
novo biosynthesis of quinolinate from tryptophan and the
subsequent synthesis of NAD and NADP from quinolinate have been
characterized either in human or in mouse (9, 10, 50-52). Several NAD
salvage routes in human have also been deduced from comparative genome analysis in combination with existing biochemical data (26). One of
these routes, the salvage of nicotinamide ribose, is directly related
to the hypothesized mechanism of tiazofurin activation (11, 19). This
mechanism includes a concerted action of ribosylnicotinamide kinase to produce NMN from nicotinamide ribose, and PNAT to convert NMN
directly to NAD. So far NAD degradation and recycling pathways are
still poorly understood. Two major types of NAD hydrolytic degradation
are known: the NAD-pyrophosphohydrolase type (producing NMN and AMP)
and the NAD-glycohydrolase type (producing nicotinamide and ADP-ribose)
(50, 51). NMN-specific activity of PNAT may be involved in the
recycling of NMN formed by the first mechanism. The second hydrolytic
reaction is directly coupled to the non-redox functions of NAD(P), such
as ADP-ribosylation, histone deacetylation, and cyclic ADP
ribose-dependent Ca2+ signaling. Nicotinamide
formed in the course of these processes can be recycled via the salvage pathways.
Relatively little is known about tissue distribution, intracellular
compartmentalization, and regulation of these multiple components of
NAD metabolism in human cells, which complicates accurate inference of
biologically relevant NAD biosynthetic routes. Clearly PNAT plays a
central role in all of these pathways. In contrast to an early dogma,
the existence of a cytosolic and mitochondria hsPNAT-3 strongly
suggests that final steps of NAD biosynthesis may occur outside of the
nucleus. All of the three characterized human PNAT isoforms display
dual substrate specificity toward either NMN or NaMN substrate and thus
are flexible to participate in either de novo or
salvage/recycling pathways via either intermediate. The nuclear
hsPNAT-1 is undoubtedly involved in the NAD regeneration in the
nucleus, but its role in NAD de novo biosynthesis is less obvious. It is tempting to speculate that hsPNAT-3 may be one of the
key participants in NAD biogenesis outside of the nucleus. Further
experiments are required to revisit the early dogma on the cellular NAD
biosynthesis, which was based solely on the localization of the major
PNAT activity inside nuclei.
Because the NAD molecule serves both as redox cofactor in many basic
cellular metabolic pathways and as substrate for several regulatory
reactions, understanding the NAD biosynthesis and how it is regulated
may be important for deciphering the link between metabolism and
various regulatory events, such as gene silencing, Ca2+
signaling, and protein ADP-ribosylation. As the central enzyme in NAD
metabolism, human PNATs appear to be subject to regulations by
phosphorylation and interaction with ADP-ribosyltransferase (24). The
identification and characterization of three distinct human PNAT
isoenzymes represent a step forward in human NAD biosynthesis studies.
Further experiments in various types of cells and in a variety of
conditions are required to characterize the respective roles of these
PNAT isoenzymes in a complex network of NAD metabolism and regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-competent
cells (Invitrogen) for protein expression. The cells were induced by
adding 0.8 mM isopropyl-1-thio-
-D-galactopyranoside when grown
to A600 = 0.5-0.6 and harvested after
shaking at 20 °C overnight. The clarified cell lysate was mixed with
the proper amount of nickel-nitrilotriacetic acid-agarose beads
(Qiagen) and incubated at 4 °C for 2 h. The bound protein was
eluted with 50 mM HEPES (pH 7.2), 300 mM NaCl, and 250 mM imidazole. After the elution, the protein was
treated with TEV protease at 4 °C overnight to cleave the
His6 tag. As the final purification step, the
tag-removed protein was passed through a Mono S ion-exchange column
(Amersham Biosciences) and eluted with a gradient of NaCl.
1cm
1 was used for rate
calculations. One unit of enzyme was defined as capable of producing 1 µmol of NADH per minute. To measure NaMNAT activity, the procedure
was modified by introducing an additional enzymatic step: conversion of
NaAD to NAD by NAD synthetase (NADS, EC 6.3.5.1) (40). 0.3 unit of NAD
synthetase from Corynebacterium glutamicum (gift from
Dr. K. Shatalin, Integrated Genomics Inc.) and 10 mM
NH4Cl were added to the same reaction mixture, and the reaction was initiated by adding 10 µl of 20 mM NaMN.
70 °C for 7 days. The
blots were subsequently washed and rehybridized with radiolabeled
-actin probe to normalize for mRNA loading levels.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Expression of hsPNAT-3 in human tissues.
The poly(A)+-RNA from human tissues were analyzed by
Northern hybridization with a 32P-labeled probe
corresponding to the coding region of hsPNAT-3. The blot was
subsequently washed and rehybridized with radiolabeled -actin to
normalize for mRNA loading levels.
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Fig. 2.
Subcellular compartmentalization of hsPNAT-2
and hsPNAT-3. A, HEK293 and HeLa cells
transfected with plasmid pEGFP-hsPNAT3 were stained with MitoTracker®
Red and examined under a confocal fluorescence microscope. The
three images show the same group of cells visualized using
different color channels. B, HeLa cells transfected with
pEGFP-hsPNAT2 and stained with MitoTracker® Red. The distribution of
the fusion protein GFP-hsPNAT2 in HEK293 cells is the same as in the
HeLa cells and is not shown.
atoms about 0.9 Å (Fig. 3A). The structure contains a
central parallel six-stranded
sheet surrounded by helices in a
typical Rossmann fold topology. A region consisting of residues
106-125 is disordered. The corresponding region in hsPNAT-1 contains
the nuclear localization signal (NLS) sequence and is also disordered.
The main differences between the structures of the two hsPNAT
isoforms are located at the loop regions between residues 52 and 57, between residues 166 and 171, and between residues 192 and 199 preceding the ISSTXXR motif (Fig. 3A).
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Fig. 3.
The overall structure of hsPNAT-3.
A, stereo view of the C trace of
hsPNAT-3 monomer (red) superimposed with hsPNAT-1
(blue). Every 20th residue is labeled. B, stereo
diagrams of hsPNAT-3 tetramer. Each monomer is colored differently. The
disordered regions are shown as dotted loops. Two orthogonal
views are shown. The bound NAD molecules are shown as
ball-and-stick representations.
atoms of the apo and various ligand-bound complexes range from 0.47 to 0.56 Å, whereas differences between ligand-bound structures are smaller, ranging from 0.24 to 0.39 Å. The
structures of the NAD- and NaAD-bound complexes are nearly identical
with an r.m.s.d. of 0.18 Å.
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Fig. 4.
Stereo diagram of superposition of the active
site between apo-form (in yellow) and NAD-bound (in
cyan) hsPNAT-3. The bound NAD (in
orange) is shown in ball-and-stick
representation. Several protein residues discussed in the text are also
shown. This and subsequent figures were generated with MOLSCRIPT (53)
and BOBSCRIPT (54) and rendered with gl_render (L. Esser, unpublished
program).
-phosphate interacts with the main-chain amide
groups of Gly-14 and Ser-15. The
-phosphate interacts with N
2 of the His-22 side-chain imidazole. This histidine residue is the invariant second histidine in the (H/T)IGH motif that has been
shown to be critical for the catalysis (16). The
-phosphate of the
ATP interacts with the side chains of Lys-56, Arg-206, and Thr-203,
with the main-chain amide of Thr-203, and with the Lys-140 side chain
from monomer B. Thr-203 and Arg-206 are part of the ISSTXXR
motif located at the N terminus of an
helix and are highly
conserved in many (T/H)IGH motif-containing nucleotidyltransferases (18). Our structures, along with the ATP-bound structures of Methanococcus jannaschii NMNAT (15) and E. coli
phosphopantetheine adenylyltransferase (PPAT) (46), showed that this
ISSTXXR motif is involved in the binding of the
-phosphate of the bound ATP and is likely to be in the position to
bind the leaving pyrophosphate group as well.
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Fig. 5.
The ATP and NMN binding sites in
hsPNAT-3. A, stereo view of the ATP binding site.
B, the Fo Fc omit
map for NMN molecule in monomer B of the NMN binary complex showing the
alternative conformations of NMN phosphate. The map is contoured at
2
. C, stereo view of the NMN binding site. The substrates
ATP and NMN and a bound sulfate molecule are shown as bonds
colored according to atom types. The two alternative conformations
of the bound NMN are shown. Relevant protein residues are shown in
ball-and-stick representation. The hydrogen bonds are
indicated by dotted lines.
-phosphate
of ATP and forms extensive interactions with the surrounding protein
atoms. Such a sulfate is also observed in the
Methanobacterium thermoautotrophicum NMNAT·NAD
complex structure (16). We have not observed a Mg2+
ion in any of the hsPNAT-3 complex structures, because no
Mg2+ is present in the crystallization buffer. It is likely
that a Mg2+ ion is needed for the catalysis (see below) but
is not required for substrate binding.
-phosphate (Fig. 5C). The
presence of this sulfate might contribute to keep the NMN in the bent conformation.
1 of
His-152. As discussed below, this water molecule may play a role in the
dual specificity of the enzyme.
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Fig. 6.
Comparison of conformations of the bound
ligands in hsPNAT3. A, the electron density of NMN and
AMP-CPP in hsPNAT-3 ternary substrate complex. The
Fo Fc omit map is contoured at
2
. Here the NMN molecule is shown to adopt exclusively the bent
conformation. B, superposition of ligands in the NMN binary
complex (yellow, both NMN conformers are shown), in the
AMP-CPP·NMN ternary complex (cyan), and in the NAD product
complex (orange). The putative Mg2+ ion binding
site (as inferred from the M. jannaschii NMNAT·ATP complex
structure) is indicated by a purple sphere.
Crystal data and refinement statistics of hsPNAT-3 crystals
-phosphate (47). The propensity of NMN to adopt an extended
conformation would bring the NMN phosphate close to the ATP
-phosphate for such an attack. The interactions of the enzyme with
ATP and NMN phosphates would help to stabilize the two negatively
charged phosphate groups in close proximity. Although the required
Mg2+ is not observed in our crystal structures, its
position may be inferred from the M. jannaschii NMNAT·ATP
complex structure, in which a Mg2+ is found to be
coordinated to the oxygens from all three ATP phosphates (15) (Fig.
6B). The location of the Mg2+ between the ATP
-phosphate and the NMN phosphate suggests that it is likely to play
a role in mediating the nucleophilic attack between the two phosphate
groups. There are extensive interactions between the enzyme and the ATP
-phosphate but few interactions with the
-phosphate of ATP (Fig.
5A). This binding mode would not only help to make the
,
-pyrophosphate a good leaving group but also favor its
rearrangement after the cleavage of the
/
-phosphodiester bond.
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ACKNOWLEDGEMENTS |
---|
We thank Leeju Wu in Steve McKnight's laboratory for help with the Northern blot analysis, Susan Sun in Helen Yin's laboratory for help with the use of fluorescence microscopes, Yingming Zhao for help with the mammalian cell culture, Hongtao Yu for helpful discussions, and Lisa Kinch for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM65243 (to H. Z.) and The Robert A. Welch Foundation Grant I-5051 (to N. V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. S1 and its legend on one text page.
The atomic coordinates and the structure factors (code 1NUP, 1NUQ, 1NUR, 1NUS, 1NUT, and 1NUU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.:
214-648-9299; Fax: 214-648-9099; E-mail: zhang@chop.swmed.edu.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M300073200
2 H. Zhang, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
NAD, nicotinamide
adenine dinucleotide;
NaAD, nicotinic acid adenine dinucleotide;
NMN, nicotinamide mononucleotide;
NaMN, nicotinic acid mononucleotide;
NMNAT, NMN adenylyltransferase;
NaMNT, NaMN adenylyltransferase;
PNAT, pyridine nucleotide adenylyltransferase;
AMP-CPP, adenosine
5'-(,
-methylene)triphosphate;
PPAT, phosphopantetheine
adenylyltransferase;
r.m.s.d., root mean square deviation;
GFP, green
fluorescent protein;
EGFP, enhanced GFP;
NLS, nuclear localization
signal;
c.c., correlation coefficient.
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