From the Division of Molecular and Structural
Biology, Ontario Cancer Institute, 610 University Avenue
Toronto M5G 2M9, Ontario, Canada, the Departments of
§ Medical Biophysics,
Biochemistry, and
Molecular and
Medical Genetics and the ¶ C. H. Best Institute, Banting and Best
Department of Medical Research, University of Toronto,
Toronto M5S 1A8, Ontario, Canada, and ** Integrative Proteomics,
Toronto M5G 1L6, Ontario, Canada
Received for publication, September 26, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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Nicotinamide mononucleotide
adenylyltransferase (NMNATase) catalyzes the linking of
NMN+ or NaMN+ with ATP, which in all
organisms is one of the common step in the synthesis of the ubiquitous
coenzyme NAD+, via both de novo and salvage
biosynthetic pathways. The structure of Methanobacterium
thermoautotrophicum NMNATase determined using multiwavelength
anomalous dispersion phasing revealed a nucleotide-binding fold
common to nucleotidyltransferase proteins. An NAD+ molecule
and a sulfate ion were bound in the active site allowing the
identification of residues involved in product binding. In addition, the role of the conserved
16HXGH19 active site motif
in catalysis was probed by mutagenic, enzymatic and crystallographic
techniques, including the characterization of an
NMN+/SO Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1)
catalyzes the synthesis of nicotinamide adenine dinucleotide (NAD+) or nicotinic acid dinucleotide
(NaAD+)1 from
nicotinamide mononucleotide (NMN+) or nicotinic acid
mononucleotide (NaMN+), respectively, by transferring the
adenylyl part of ATP and concomitantly releasing pyrophosphate
(PPi) (Fig. 1A).
The reaction product, NAD+, plays a central role in many
cellular processes; it functions as a coenzyme in reduction-oxidation
reactions and as a substrate in DNA ligation and protein
ADP-ribosylation reactions (1). There is also considerable medical
interest in this enzyme as it is implicated in the metabolism of the
antitumor drug tiazofurin (2). In vivo, tiazofurin is
phosphorylated to tiazofurin monophosphate and then converted by
NMNATase to the actual pharmacophore thiazole-4-carboxamide adenine
dinucleotide, an analog of NAD+. Thiazole-4-carboxamide
adenine dinucleotide is a potent inhibitor of inosine monophosphate
dehydrogenase causing arrest of guanylate biosynthesis and thus
inhibition of tumor cell proliferation. Consistent with this
interpretation, low NMNATase activity is observed in cancer patients
showing resistance to tiazofurin therapy (2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, reaction catalyzed by NMNATase
resulting in the synthesis of NAD+. B, amino
acid sequence alignment of archaeal and bacterial NMNATase proteins
from M. thermoautotrophicum (Mth), M. jannaschi (Mja), Methanolobus tindarius
(Mti), Archaeoglobus fulgidus (Afu),
Pyrococcus abyssi (Pab), Aquifex
pernix (Ape), E. coli (Eco),
Salmonella typhymurium (Sty), Hemophilus
influenza (Hin), Strepatomyces
viridochromogenes (Svi), Mycobacterium
tuberculosis (Mtu), and Synechocystis sp.
(Ssp). Residues that are conserved between bacterial as well
as archaeal organisms are highlighted in black. Residues
that are only conserved between archaeal organisms and are involved in
ligand binding are highlighted in gray.
Two mechanisms for the NMNATase-catalyzed synthesis of NAD+
have been postulated; the first one assumes a double displacement reaction that involves the formation of an adenylyl enzyme covalent intermediate upon release of pyrophosphate followed by transfer of the
adenylyl group to NMN+ to form NAD+, whereas
the second one describes a nucleophilic attack of the 5'-phosphate of
NMN+ on the -phosphate of ATP to form NAD+
and releasing PPi. The latter mechanism is supported by
17O NMR studies of NAD+ synthesis (3), but a
complete understanding of the catalytic chemistry awaited more detailed
structural information.
NMNATase proteins have been identified, purified, and characterized from archaea, bacteria, and eukarya. All of these proteins are oligomeric; trimeric, tetrameric, and hexameric forms have been observed (1). Several NMNATase genes have been sequenced from a variety of sources (Fig. 1B). Although these gene products remain annotated in the GenBankTM data base as of unknown function, related sequences from Methanococcus jannaschii, Escherichia coli, Synechocystis sp., and Sulfolobus solfataricus have been overexpressed as recombinant proteins in E. coli and shown to exhibit NMNATase activity (4-8).
Recently, the crystal structure of NMNATase from M. jannaschii in complex with ATP and Mg2+ was reported
(9). Our results on the NAD+ and NMN+ complexes
of the Methanobacterium thermoautotrophicum enzyme complement this result, especially when interpreting the catalytic mechanism of NMNATase. We describe the crystal structures of the NAD+ complex of NMNATase and of the NMN+
complex of an active site mutant (H19A) of NMNATase at 1.9 and 2.5 Å resolution, respectively. These structural results, combined with
mutagenesis and enzymatic experiments, define the spatial geometry of
the ligand binding sites, identify residues with potential roles in
substrate binding and catalysis, and suggest aspects of the product
release mechanism.
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EXPERIMENTAL PROCEDURES |
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Cloning, Protein Expression, and Purification--
The NMNATase
gene (GenBankTM accession number AE000803) was
amplified by polymerase chain reaction using M. thermoautotrophicum genomic DNA and cloned into the pET15b
(Novagen) expression vector at the NdeI and BglII
sites. Recombinant NMNATase was overexpressed in E. coli
BL21 Gold (DE3) cells (Stratagene) harboring a plasmid encoding rare
E. coli tRNA genes. The cells were grown at 37 °C in
Luria-Bertoni broth with carbenicillin (50 µg/ml) and kanamycin (50 µg/ml) to an A600 nm of 0.7 and
induced overnight with 0.5 mM
isopropyl--D-thiogalactopyranoside at 24 °C. The
bacteria were harvested by centrifugation and resuspended in binding
buffer (50 mM Tris, 500 mM NaCl, 5% glycerol,
and 5 mM imidazole) supplemented with 2 mM
phenylmethylsulfonyl fluoride. Bacteria were lysed by several passages
through a French pressure cell at 1.4 × 108 pascals,
and DNA was sheared by sonication. Cell debris was removed through
centrifugation for 30 min at 20,000 × g. Contaminating E. coli proteins were removed by heating for 20 min at
55 °C followed by centrifugation at 5000 × g for 30 min. The supernatant was applied by gravity to a DE52 column (Whatman)
immediately coupled to a Ni2+ column (Qiagen). The
Ni2+ column was washed with 50 volumes of binding buffer
containing 30 mM imidazole. The bound NMNATase was eluted
from the Ni2+ column with 500 mM imidazole in
binding buffer. The hexahistidine tag was cleaved by digesting for
16 h with thrombin (1 µg of thrombin per mg of recombinant
protein) at 4 °C in binding buffer made 2.5 mM in
CaCl2. NMNATase was then dialyzed against 500 mM NaCl in 10 mM HEPES (pH 7.5) and
concentrated to 10 mg/ml using BioMax concentrators (Millipore). Mutant
H19A NMNATase was purified as described above for WT NMNATase.
For the preparation of selenomethionine (Se-Met)-enriched
protein, NMNATase was expressed in a methionine auxotroph strain B834(DE3) of E. coli (Novagen) and purified under the same
conditions as native NMNATase with the addition of 5 mM
-mercaptoethanol in all buffers.
Crystallization-- Screening for crystallization conditions was performed using Hampton Research Crystal Screens I and II at room temperature in VDX plates with the hanging drop vapor diffusion method. 2 µl of protein solution (10 mg/ml) were mixed with 2 µl of the various reservoir solutions and equilibrated with 500 µl of this solution. Crystals in the form of hexagonal rods appeared after 24 h in crystallization set-ups containing ammonium sulfate or lithium sulfate as precipitant. Crystals selected for native and multiwavelength anomalous dispersion (MAD) data collection were grown in 1.5 M LiSO4 and 100 mM HEPES at pH 7.5 at 20 °C. Crystals of H19A NMNATase were grown in 1.6 M LiSO4 and 100 mM HEPES at pH 7.5 at 20 °C.
X-ray Diffraction and Structure Determination--
Making use of
the anomalous scattering of selenium atoms, a three-wavelength
MAD experiment was carried out at 100 K on beamline BM14D, APS, using a
Q1 CCD detector (Area Detector Systems Corp.). The crystal was
flash-frozen with crystallization buffer plus 30% glycerol as
cryoprotectant. Diffraction data from native crystals of WT and H19A
NMNATase were collected on beamline BM14C, APS, at 100 K using a Q4 CCD
detector (ADSC). Both MAD and native data were processed and scaled
with the DENZO/SCALEPACK suite of programs (10). The selenium sites
were located using SOLVE (11) and refined using SHARP (12). The
electron density map was improved using Density Modification
from the CCP4 package (13). Model building was done with O (14),
and CNS (crystallography and NMR system) (15) was used for
refinement. Water molecules were initially picked using CNS and then
manually verified in O using the following criteria: a peak of at least
2.5 in an Fo
Fc map, a peak of at least 1.0
in a 2Fo
Fc map, and reasonable intermolecular interactions.
Crystallographic and refinement statistics are found in Tables
I and II,
respectively. The programs MOLSCRIPT (16), RASTER 3D (17), SPOCK (18),
and LIGPLOT (19) were used in the production of the figures.
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Gel Filtration-- Gel filtration of NMNATase was performed with a Superdex 200 prep 16/60 (Amersham Pharmacia Biotech) column equilibrated with 10 mM HEPES and 500 mM NaCl using high pressure liquid chromatography (LKB-Wallac). Protein standards included aldolase, bovine serum albumin, ovalbumin, and cytochrome c. Chromatography was performed at 4 °C at a flow rate of 0.5 ml/min.
Site-directed Mutagenesis-- Site-directed mutagenesis to change His-19 to Ala was carried out using QuikChangeTM (Stratagene). The mutagenic primers were 5'-CACAGGGGCGCACTGCAGGTC and 5'-GACCTGCAGTGCGCCCCTGTG for H19A. DNA encoding WT NMNATase cloned into pET-15B was used as template for the polymerase chain reaction mutagenesis reaction. Briefly, 25 ng of template DNA was incubated with the appropriate mutagenic primers, dNTPs, and Pfu DNA polymerase using the cycling parameters recommended in the supplier's manual. Following the temperature cycling step, DpnI was added to each amplification reaction and incubated at 37 °C for 6 h followed by transformation of the mutagenized plasmid into XL2 Blue cells.
Enzymatic Assay--
WT NMNATase activity was measured in a
coupled assay according to Raffaelli et al. (6). Varying
amounts of NMNATase (1-1000 ng) were incubated with 2 mM
NMN+, 2 mM ATP, 10 mM
MgCl2, and 50 mM HEPES (pH 7.5) at 65 °C for 20 min. The amount of NAD+ formed was measured
spectrophotometrically at 340 nm using alcohol dehydrogenase to convert
NAD+ to NADH. The enzymatic activity of H19A NMNATase was
measured the same way but varying the amount of enzyme in each assay
from 1 to 5000 µg.
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RESULTS AND DISCUSSION |
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Structure Determination--
The structure of the NAD+
complex of NMNATase has been determined by the MAD method using
selenium as the anomalous scatterer. The resulting electron density is
of high quality except for the loop consisting of residues 124-129, in
which the main chain density is continuous but increased mobility has
compromised the clarity of side chain densities. In addition, 12 C-terminal and 3 N-terminal amino acids are not visible in the electron
density map. The final model contains 167 amino acids (residues
4-171), with Pro-14 in a cis-conformation; 119 water
molecules; one molecule of NAD+; 1 sodium; and 1 sulfate
ion (Fig. 2A). Refinement at
1.9 Å resolution resulted in an Rcryst of 0.212 and an Rfree of 0.242. According to PROCHECK
(20), 92% of the residues are in the most favored regions, and no
residue is in the disallowed regions of the Ramachandran plot.
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The structure of the NMN+ complex of H19A NMNATase was determined by molecular replacement techniques. Its electron density map is of good quality except for, again, the loop including residues 124 and 129, for which no continuous electron density is observed at all. The final model of this complex contains 161 amino acids (residues 4-123 and 130-170), with Pro-14 still adopting a cis-conformation; 10 water molecules; 1 molecule of NMN+; and 1 sulfate ion. Refinement at 2.5 Å resolution gave an Rcryst of 0.236 and an Rfree of 0.294. 94.1% of the residues are in the most favored regions, and no residue is in the disallowed regions.
Overview of the Structure--
Consistent with gel filtration
studies, which for M. thermoautotrophicum NMNATase point to
a hexamer as the functional unit in solution (data not shown), the
322 symmetry of NMNATase crystals combines the single monomers
occupying each asymmetric unit into a hexameric arrangement (Fig.
2B). Each hexamer is created by two trimers rotated by
180° and layered on top of each other, with overall dimensions of
approximately 80 × 80 × 60 Å. Each subunit consists of two
domains (Fig. 2A), the first of which is the
dinucleotide-binding domain and comprises residues 4-130 with a
topological arrangement of alternating -strands and
-helices. The
twisted
-sheet at the core of the subunit consists of five parallel
-strands with topology 3-2-1-4-5 (Fig. 2A). The second domain (residues 131-170) is made up from three
-helices (helices 5-7) and is the major contributor to intratrimer subunit interactions.
Subunit Interactions-- The intratrimer interactions are almost exclusively electrostatic and occur preferentially between helix 5 of subunit A and helix 7 of subunit B. Salt bridges are formed by Arg-110A and Glu-164B; Glu-114A with His-44B and Arg-165B; and Gln-109A with His-168B. These contacts are repeated between subunits B and C as well as subunits C and A (Fig. 2B). In each case, 1457 Å2 of a total of 16,938 Å2 of molecular surface are buried upon oligomerization.
In contrast to the intratrimer interactions, the dominant intertrimer
contacts are nonpolar and hydrophobic. The side chains of -strand 3 (Ile-75, Ile-76, and Val-78) of subunit A pack against their
counterparts of
-strand 3 of subunit D with the same interactions repeated between subunits B and E, as well as C and F. In each case,
2796 Å2 of a total of 15,609 Å2 of molecular
surface are buried (Fig. 2B).
NAD+ and Sulfate Binding to WT NMNATase--
The
active site is located in a deep cleft facing a narrow channel running
along the 3-fold symmetry axis of the hexamer. The site is open to
solvent, possibly reflecting its readiness to release the product
NAD+, which, together with a sulfate ion, could easily be
identified in the electron density after the first round of refinement
(Fig. 3A). As no
NAD+ was added during enzyme purification and
crystallization, thermophilic NMNATase must have trapped its product.
NAD+ binds in an extended conformation with its adenine
ring adopting an anti orientation and both the adenylyl and
the nicotinamide ribose rings showing 3'-endo puckering (Fig.
3C). In contrast, the adenylyl ribose ring seems to be in
the 2'-endo conformation in the M. jannaschii NMNATase
structure (9). The exocyclic nitrogen of adenine is H-bonded to the
main chain carbonyls of Phe-125 and Tyr-130, two aromatic amino acids
the side chains of which interact closely. N1 binds to the main chain
amide of Phe-125 and N7 to a water molecule. The adenylyl ribose forms an H-bond (3.1 Å long) between its 3'-hydroxyl and the backbone amide
of Gly-104, in contrast to what has been found in
NAD(P)+-dependent dehydrogenases, in which the
adenylyl ribose ring is held in place by a conserved aspartate located
at the C terminus of the second -strand (21). In NMNATase, it is the
nicotinamide ribose in which one finds H-bonds between its 2'-hydroxyl
and the carboxyl side chain of Asp-80; the 3'-hydroxyl interacts with the side chain of Ser-39. The nicotinamide ring stacks with the aromatic ring of Trp-87 and its amide substituent links to the main
chain of Ile-81, the oxygen to its amino and the nitrogen to its
carbonyl atoms. In addition, the side chain of Asn-84 holds the amide
NH2 of the substituent in place (Fig. 3, C and
D). As the enzyme has to accept both NMN+ and
NaMN+ as substrates, an amide side chain is ideal for this
purpose. A single bond rotation will provide an H-bonding partner for
nitrogen as well as oxygen atoms. A flip of the Ile-81/Glu-82 peptide
bond could easily provide the proper interaction for the second oxygen atom in the carboxylate of NaMN+. Such a change in backbone
conformation would not be difficult to achieve as this region of the
chain runs along the surface of the protein molecule and seems
unconstrained.
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The "NMN-phosphate" in the pyrophosphate linkage forms H-bonds to
Asn-105. The oxygen atom bridging the two phosphorous atoms of the
pyrophosphate group contacts the main chain amide of Arg-11. The
"AMP-phosphate" binds to His-19, the main chain of Met-12, two
water molecules and a rather high (3) spherical electron density
that we interpret as a Na+ ion. This Na+ ion
could contribute to balancing the negative charges of the pyrophosphate
and of another electron-dense feature, the center of which is located
5.3 Å from the phosphorous atom of the AMP-phosphate; the shape and
position of this feature indicate a sulfate ion, an excellent
mimic of a phosphate group. Both sodium and sulfate ions could have
been provided by the crystallization buffer (0.5 M NaCl,
1.5 M Li2SO4). The two waters
mentioned above and the metal ion are located so they can bridge oxygen
atoms from the AMP-phosphate and the
SO
-phosphate of the substrate ATP and is reminiscent of the sulfate ion bound at the
-phosphate position in
the active site of glutaminyl tRNA synthetase complexed with AMP (22).
This assignment gains further credibility by interpreting the figures
portraying the binding of Mg2+-ATP to M. jannaschii NMNATase (9).
Functional and Structural Comparisons--
Analysis of the
structure of NMNATase using the program DALI (23) identified several
proteins all of them belonging to the nucleotidyltransferase
superfamily of dinucleotide-binding fold containing /
phosphodiesterases (24). Presently known members of this family are
CTP:glycerol-3-phosphate cytidyltransferase (CTP:G3PCase) (25),
glutaminyl tRNA synthetase (26), tyrosyl tRNA synthetase (27) and
phosphopantetheine adenylyltransferase (PPATase) (24). Their overall
structures are remarkably similar (r.m.s.d. of 116, 137, 124, and 143 C
atoms, equal to 2.5, 3.5, 3.9, and 2.3 Å,
respectively). PPATase not only closely resembles the overall fold of
NMNATase but even forms a hexamer as the functional unit. Nevertheless,
the arrangement of subunits relative to each other is quite different.
All of these related proteins contain a nucleotide-binding domain,
present the active site sequence motif (T/H)XGH, and
catalyze a nucleotidyltransfer reaction that is similar to that of
NMNATase. This reaction involves the attack of a nucleophilic group of
one substrate at the
-phosphate of the nucleoside triphosphate (the
second substrate), thereby forming a new phosphodiester bond and
releasing a pyrophosphate molecule.
Originally, identification of NAD+ in the active site cleft
and the DALI results indicating nucleotidyltransferase function led us
to propose that protein MT0150, annotated as "conserved" in the
M. thermoautotrophicum genome sequence data base (28), was
in fact an NMNATase. Preliminary enzymatic assays confirmed that the
enzyme catalyzed the biosynthesis of NAD+ from
NMN+ and ATP (Fig.
4B). The validity of this assignment was established when a
literature search revealed that the homologous enzyme from M. jannaschii had NMNATase activity (4, 6).
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In addition to the common nucleotide-binding fold, all of these enzymes
contain an active site sequence motif, (T/H)XGH, that is
absolutely conserved in all known archaeal and bacterial NMNATases (16HRGH19 in M. thermoautotrophicum
NMNATase; Fig. 1B). This active site sequence motif has also
been identified in other nucleotide binding enzymes catalyzing
adenylyltransferase reactions such as the ATP sulfurylase domain of
human phosphoadenosine phosphosulfate synthase (425HNGH428), A. thaliana ATP
sulfurylase, and E. coli flavin adenine dinucleotide synthetase (29HRGH32) (29-31). For several of
these enzymes, the roles of the two conserved histidine residues in
this motif have been investigated using crystallographic and
site-directed mutagenesis techniques (22, 24-27, 32). Although their
mutation to alanine residues in CTP:G3PCase, phosphosulfate synthase,
and tyrosyl tRNA synthetase demonstrated that they were essential for
catalysis (28, 32, 33), structural studies of glutaminyl tRNA
synthetase were more specific, implicating them in the binding of the
- and
-phosphates of ATP and the stabilization of the postulated
pentacoordinate transition state (22). Mutagenesis of each of the two
His residues (His-425 and His-428) to alanine in phosphosulfate
synthase and measurement of catalytic activities in crude extracts of
transformed E. coli cells confirmed that the mutant proteins
exhibited only background levels of adenylyltransferase activity
(29).
Whereas the binding sites of the nucleoside triphosphate substrates are
well characterized and overlap quite closely for all structurally known
members of the nucleotidyltransferase superfamily, PPATase is the only
one for which the interactions with the product have been described
(24). This enzyme is also the one with the highest degree of structural
similarity to NMNATase. When NMNATase and PPATase are superimposed
using their (T/H)XGH motifs, it is evident that not only do
the adenylyl moieties line up closely but the remaining chemically
dissimilar halves of the products (NMN+ versus
4'-phosphopantetheine) occupy overlapping parts of the respective
active sites, too (Fig. 4C). It is interesting to note that
space that is filled by the terminal -mercaptoethylamine moiety of
dephospho-coenzyme A in PPATase is taken up by the side chain of Trp-87
in NMNATase, which, through its large aromatic ring, provides the
nicotinamide ring with the opportunity to undergo a stacking interaction.
Mutagenic, Enzymatic, and Crystallographic Studies of His-19 in the
16HRGH19 Motif--
Superimposing the
(T/H)XGH structural motif of CTP:G3PCase, glutaminyl tRNA
synthetase, and PPATase on that of NMNATase places the respective
histidines in comparable positions, from which they bind to the -
and
-phosphate oxygens of ATP (Fig. 4A).
The importance of His-19 in NMNATase activity was investigated further using site-directed mutagenesis. Preliminary enzymatic analysis shows that the H19A mutant retains less than 1% of the specific activity of WT NMNATase, indicating a strong involvement in catalysis for this residue (Fig. 4B). The results are also in good agreement with previous studies of phosphosulfate synthase and CTP:G3PCase, in which mutagenesis of the His residues to Ala resulted in enzymes with activities lowered by at least 4 orders of magnitude (29, 34). To confirm that the H19A protein was stable and properly folded and that its lack of NMNATase activity was not caused by partial denaturation or misfolding, its crystal structure was determined.
The mutant enzyme crystallized in the same crystal form as WT NMNATase;
its polypeptide structure was quite similar to that of WT NMNATase
(r.m.s.d. of all C positions is 0.21 Å). Instead of the product
NAD+, however, a molecule of the substrate NMN+
was trapped in its active site (Fig. 3B). This finding
supports the kinetic experiments that showed a lack of NMNATase
activity for this mutant. At this point, however, we cannot distinguish between a drastic loss of affinity for ATP and an incompatibility to
undergo the chemistry necessary for catalysis. The variation in ligands
seems also to be responsible for the only remarkable structural
difference evident between the protein parts of the native and mutant
complexes. In WT NMNATase, although the loop region between residues
123 and 130 shows increased B-factors of both the main and side chains
indicating flexibility, there is clear density to follow the main
chain. For residues 124-129 in the H19A mutant, however, there is no
continuous electron density visible at all. Obviously, the presence of
the adenine ring of ATP or NAD+ is necessary to at least
partially stabilize this stretch of peptide chain.
Proposed Reaction Mechanism--
Several conserved and positively
charged residues (Arg-11, His-16, His-19, and Arg-136) line the active
site of NMNATase, and their interactions with ATP have recently been
determined. The absence of any covalent adenylyl-NMNATase intermediate
has also been established (9). When the position of ATP is considered together with the results of the crystal structure of H19A NMNATase complexed with NMN+, it becomes clear that NMN+
is bound in such a way that its phosphate group is positioned close to
the -phosphate of ATP, ready to attack from the side opposite of the
pyrophosphate leaving group, in line with proposed direct attack
adenylyltransferase mechanisms (22). A small shift in position
accompanies the transition of the
-phosphate of ATP to the
AMP-phosphate of NAD+. This seems to be reflected in a
switch of the pucker of the adenosine ribose from 3'-endo to 2'-endo.
The crystallographic results are fully consistent with 17O
NMR studies of NAD+ that support direct attack of ATP by
NMN+ (3). As the phosphate group of NMN+ is a
reasonable nucleophile and pyrophosphate a good leaving group, residues
from the active site of NMNATase may not be required to directly
participate in the chemistry of the reaction by acid/base or covalent
catalysis (22, 29). Locking the reaction partners into the proper
geometry and electrostatic support might be all that is needed
for catalysis to occur. An equivalent adenylyltransferase mechanism was
first reported for tRNA aminoacylation by glutaminyl tRNA synthetase
(22). We believe that this is the mechanism used by the archaeal and
bacterial NMNATases because the HXGH active site motif is
conserved at an equivalent position among all prokaryotic organisms for
which the corresponding gene has been sequenced (Fig. 1B).
The existence of Mg2+-ATP (9) and NMN+
complexes (see above)2
shows that archaeal NMNATases have the capacity to bind the two substrates independently from each other, whereas eukaryotic enzymes follow an ordered bi-bi mechanism (1).
Proposed Product Release Mechanism-- When M. thermoautotrophicum NMNATase is expressed in E. coli, NAD+ molecules are trapped within the active site of the protein. Full catalytic function of this enzyme is only observed at 65 °C, the optimal growth temperature of the archaeal thermophilic source. NAD+ bound to WT NMNATase could only be released if the protein sample was heated to 65 °C (data not shown). These results point to an obligatory conformational change in this protein before the release of product can occur.
Before the binding of the ATP substrate, the loop surrounding the
adenine ring (residues 123-130) is quite mobile, as indicated by the
absence of reasonable corresponding electron density in the
NMN+ complex of H19A NMNATase. D'Angelo et al.
(9) showed that Arg-121 in M. jannaschii NMNATase,
which corresponds to Arg-127 in the M. thermoautotrophicum
enzyme, is bound to one of the oxygens of the -phosphate of ATP. Its
aliphatic chain undergoes hydrophobic interactions with the adenine
ring moiety (9). Once NAD+ is generated, however, this
tighter interaction seems to loosen up again, resulting in higher
B-factors for main chain atoms and weaker density at the positions of
the side chains as seen in our structure of the NAD+-WT
NMNATase complex. Despite this increased mobility, it is clear that in
the NAD+-WT NMNATase complex Arg-127 is pointing away from
the adenine ring moiety (Fig. 3C). Its guanidinium group is
now ~15 Å from where it is positioned in the ATP-Mg2+
NMNATase complex. We presume that at room temperature the thermal energy is not sufficient to break the main chain away from the adenine
ring keeping NAD+ bound at the active site. At elevated
temperatures, however, the loop could separate from the adenine ring
moiety as the first step in product release.
M. thermoautotrophicum NMNATase is only one example of a
surprising number of proteins from thermophilic organisms seen to carry
ligands with them when purified from overexpressing E. coli cells (35, 36). If this effect can be verified for more cases it could
convey additional advantages to the use of thermophilic proteins in the
challenging task of assigning function to hitherto unknown parts of proteomes.
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ACKNOWLEDGEMENTS |
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We thank the staff of BioCARS for their help during data collection at Sector 14 of the Advanced Photon Source. V. S. thanks Cristofer Enroth and Ulug Ünligil for their support during structure determination.
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FOOTNOTES |
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* Use of the Advanced Photon Source was supported by the Basic Energy Sciences, Office of Science, United States Department of Energy, under Contract W-31-109-Eng-38. Use of the BioCARS Sector 14 was supported by the National Center for Research Resources, National Institutes of Health, under Grant RR07707.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 atomic coordinates and the structure factors (code 1ej2 for WT NMNATase and code 1hyb for H19A NMNATase) 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: Division of Molecular and Structural Biology, Ontario Cancer Institute, 610 University Ave., Toronto M5G 2M9, Ontario, Canada. E-mail: pai@hera.med.utoronto.ca.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M008810200
2 V. Saridakis, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: NaAD+, nicotinic acid dinucleotide; MAD, multiwavelength anomalous dispersion; NMNATase, nicotinamide mononucleotide adenylyltransferase; WT, wild type; G3PCase, glycerol-3-phosphate cytidyltransferase; PPATase, phosphopantetheine adenylyltransferase; NaMN+, nicotinic acid mononucleotide; r.m.s.d., root mean square deviation.
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