Mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from Enterococcus faecalis

Article abstract of DOI:10.1074/jbc.M113.524207

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPsregulatetheenzyme activity was not defined.Here, wepresent the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.

Full text of DOI:10.1074/jbc.M113.524207

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 5, pp. 2815–2824, January 31, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Mechanisms of Allosteric Activation and Inhibition of the
Deoxyribonucleoside Triphosphate Triphosphohydrolase
ꢀ
□
S
from Enterococcus faecalis*
Received for publication, October 2, 2013, and in revised form, November 29, 2013 Published, JBC Papers in Press, December 12, 2013, DOI 10.1074/jbc.M113.524207
Ivan I. Vorontsov‡1, Ying Wu§1, Maria DeLucia§, George Minasov‡, Jennifer Mehrens§, Ludmilla Shuvalova‡,
Wayne F. Anderson‡2, and Jinwoo Ahn§3
From the ‡Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of
Medicine, Chicago, Illinois 60611 and the §Department of Structural Biology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15260
Background: EF1143 from Enterococcus faecalis is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase.
Results: Enzymatic studies and crystal structure of EF1143 bound to dNTPs show that dNTP binding at two adjacent allosteric
sites can differentially modulate enzyme catalysis.
Conclusion: EF1143 enzyme catalysis is differentially regulated by four dNTPs.
Significance: Learning how the enzyme activity is regulated by substrates is fundamental for understanding enzyme catalysis
and allostery.
EF1143 from Enterococcus faecalis, a life-threatening pathogen
that is resistant to common antibiotics, is a homo-tetrameric
deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase
DNA replication requires an appropriate balance of deoxyri-
bonucleoside triphosphate (dNTP)4 pools to minimize muta-
tions (1–3). The cellular levels of four canonical dNTPs (dATP,
(dNTPase), converting dNTPs into the deoxyribonucleosides and dGTP, dCTP, and dTTP) are tightly controlled by ribonucleo-
side diphosphate reductases and dNTP triphosphohydrolases
(dNTPases) (4, 5). The former converts ribonucleoside diphos-
phate or ribonucleoside triphosphate (NTP) to deoxyribo-
nucleoside diphosphate or dNTP (4), and the latter hydrolyzes
dNTP into deoxyribonucleoside (dN) and triphosphate (6, 7).
The dNTPases also play an important role in the cellular
defense against pathogens. In primates, a dGTP-activated
dNTPase, sterile alpha motif (SAM) and histidine-aspartate
(HD) domain-containing protein 1 (SAMHD1), maintains
dNTP pools at low levels, thereby blocking reverse transcrip-
tion of retroviruses, including HIV and simian immunodefi-
ciency virus, in some cell types (8–13). The virulence factor
Vpx in HIV-2 and simian immunodeficiency virus antagonizes
SAMHD1 by directing it for proteasome-dependent degrada-
tion (9, 10, 14). Similar host dNTPase-pathogen protein antag-
onist interplay is also observed in prokaryotes; the gene 1.2
product, encoded by bacteriophage T7, inhibits the Escherichia
coli dGTPase, allowing productive infection (15, 16).
triphosphate. The dNTPase activity of EF1143 is regulated by
canonical dNTPs, which simultaneously act as substrates and
activity modulators. Previous crystal structures of apo-EF1143 and
the protein bound to both dGTP and dATP suggested allosteric
regulation of its enzymatic activity by dGTP binding at four iden-
tical allosteric sites. However, whether and how other canonical
dNTPsregulatetheenzymeactivitywasnotdefined. Here, wepres-
ent the crystal structure of EF1143 in complex with dGTP and
dTTP. The new structure reveals that the tetrameric EF1143 con-
tains four additional secondary allosteric sites adjacent to the
previously identified dGTP-binding primary regulatory sites.
Structural and enzyme kinetic studies indicate that dGTP
binding to the first allosteric site, with nanomolar affinity, is
a prerequisite for substrate docking and hydrolysis. Then, the
presence of a particular dNTP in the second site either
enhances or inhibits the dNTPase activity of EF1143. Our
results provide the first mechanistic insight into dNTP-me-
diated regulation of dNTPase activity.
Enterococcus faecalis is a Gram-positive bacterium found in
the intestinal tract of humans. It is a common, life-threatening,
hospital-acquired infection, due to frequent mutations that
result in the development of antibiotic resistance (17–20).
Understanding the molecular mechanisms of E. faecalis patho-
genicity and adaptation to antibiotics is important to prevent
disease. Several virulence factors encoded by Enterococcus have
been identified (21, 22). In this study, we focused on a dNTPase,
which controls dNTP pools in the organism.
* This work was supported in part by National Institutes of Health Grant U54
GM094585 for the Midwest Center for Structural Genomics (to W. F. A.) and
Grant P50GM082251 (to J. A.). This work was also supported by funds from
the University of Pittsburgh School of Medicine (to J. A.).
ꢀ This article was selected as a Paper of the Week.
□S
This article contains supplemental Figs. S1–S5.
Theatomiccoordinatesandstructurefactors(code 4LRL)havebeendepositedin
the Protein Data Bank (http://wwpdb.org/).
1 Both authors contributed equally to this work.
2 To whom correspondence may be addressed. Tel.: 312-503-1697; Fax: 312- 4 The abbreviations used are: dNTP, deoxyribonucleoside triphosphate;
503-5359; E-mail: wf-anderson@northwestern.edu.
3 To whom correspondence may be addressed. Tel.: 412-383-6933; Fax: 412-
648-9008; E-mail: jahn@structbio.pitt.edu.
dNTPase, deoxyribonucleoside triphosphate triphosphohydrolase; dATPase,
dATPtriphosphohydrolase;SAMHD1, sterilealphamotifandHDdomain-con-
taining protein 1; HD domain, histidine-aspartate domain.
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Allosteric Activation and Inhibition of EF1143 dNTPase
TABLE 1
Biophysical characterization of several dNTPases indicates
that they exist as homo-oligomers. For example, crystal struc-
tures of the HD domains of human SAMHD1 and bacterial
EF1143, a dNTPase from E. faecalis, showed that they are dimer
and tetramer, respectively (Protein Data Bank (PDB) IDs 3U1N
and 3IRH, respectively) (6, 23). Similarly, 067745_AQUAE, a
dNTPase from the prokaryotic organism Aquifex aeolicus, is
thought to be active as a tetramer (24), whereas Tt-dNTPase
from Thermus thermophilus HB8 is a hexamer (25), based on
crystal structures (PDB IDs 2HEK and 2DQB, respectively).
The nature of the homo-oligomeric quaternary states of
dNTPases suggests allosteric regulation of their enzymatic activity
byeffector molecules. Indeed, SAMHD1undergoesdGTP-depen-
dent tetramerization, which is the catalytically active form of the
enzyme (26, 27), and extensive kinetic characterization of the Tt-
dNTPase showed that its enzymatic activity was regulated by both
dATP and dCTP (28). The most intriguing feature of the biochem-
ically characterized dNTPases, including SAMHD1, Tt-dNTPase,
andEF1143, isthatdNTPsarebotheffectorsandsubstrates. Struc-
tural comparison of the apo form of EF1143 with a dGTP effector
and dATP substrate-bound form uncovered a few important fea-
tures of its allosteric regulation and catalytic activity. Still, many
mechanistic aspects of its complex regulation remained unclear.
Here, we present the crystal structure of EF1143 in complex with
dGTP and dTTP, bound at two distinctive regulatory sites. The
residues at the primary allosteric site are configured to selectively
bind only dGTP with high affinity. Detailed biochemical analyses,
guided by the crystal structure, suggest that dTTP and dGTP func-
tion as negative regulators of the enzyme, whereas dATP and
dCTP are positive allosteric effectors, binding to the same pro-
miscuous secondary regulatory site adjacent to the dGTP-bind-
ing primary allosteric site. Taken together, these results reveal
complex molecular mechanisms of substrate-mediated regula-
tion of EF1143 catalysis.
DatacollectionandrefinementstatisticsfortheE. faecalisEF1143pro-
tein complex with Ni2؉, dTTP, and dGTP
r.m.s.d., root mean square deviation.
Complex with Ni2؉/dGTP/dTTP
Data collection
Space group
P212121
a, b, c (Å)
91.54, 144.63, 155.56
81,766
Unique reflection
Test set
4304
Resolution/last shell (Å)
I/␴(I)
30-2.35/2.43-2.35
16.8/2.1
Rmergea (%)
0.07/0.43
99.4/94.9
4.2/2.6
Completeness (%)
Overall redundancy
Refinement
b
Rwork
0.184
c
Rfree
0.235
B
iso (protein/ligands/water), Å2
46.1/43.9/46.4
0.009
r.m.s.d. bond length, Å2
r.m.s.d. bond angle, °
Ramachandran distribution
Most favored
1.15
1482 (91.8%)
129 (8.0%)
4 (0.2%)
734
Allowed
Outside allowed
Water molecules
PDB ID
4LRL
a Rmerge ϭ ΑhΑi͉Ih,i Ϫ ͗Ih͉͘/ΑhΑiIh,i, where Ih,i is the ith observation of the reflec-
tion h, whereas ͗Ih͘ is the mean intensity of reflections h.
b Rwork ϭ ΑʈFo͉Ϫ ͉Fcʈ/͉Fo͉.
c Rfree was calculated with a small fraction (5%) of randomly selected reflections.
22 °C. The crystals were flash-frozen in liquid nitrogen. Diffrac-
tion data were collected at 100 K at the LS-CAT (21-ID-F)
beamline at the Advanced Photon Source, Argonne National
Laboratory. All the data were processed with HKL2000 (30).
Structure Determination and Sequence Analysis—The struc-
ture of the EF1143 in complex with dGTP and dTTP was solved
by molecular replacement using Phaser (31) and chain A from
the EF1143-dGTP-dATP complex as a search model (PDB ID
3IRH). The structure was completed using Coot (32) and
REFMAC (33). Crystallographic data and the refinement statis-
tics are in Table 1. Graphical presentations were done with
PyMOL (41). Sequence homology analysis was performed using
BLAST (34) and ClustalW (35).
EXPERIMENTAL PROCEDURES
Protein Expression and Purification—Cloning of N-termi-
nally His6-tagged E. faecalis HD-domain protein EF1143 was
described previously (23). Site-specific mutants of EF1143 were
prepared using QuikChange mutagenesis kits (Agilent). Wild-
type and mutant EF1143 proteins were expressed in Ros2 (DE3)
using auto-induction medium at 18 °C for 24 h (29). The recom-
binant proteins were purified by two-step liquid column chroma-
tography: a 5-ml nickel-nitrilotriacetic acid column (GE Health-
care) and a Hi-Load Superdex200 16/60 column. Purified proteins
were stored in a gel filtration column chromatography buffer con-
taining 25 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1 mM
DTT, 10% glycerol, and 0.02% sodium azide.
dNTPase Assays—EF1143 enzyme activities were studied in a
reaction buffer containing 20 mM Tris-HCl, pH 7.8, 50 mM
NaCl, 2 mM MgCl2, 5% glycerol, and varying concentrations of
dNTPs, as indicated in the figure legends, with 0.1 ␮M EF1143
and 5 ␮M BSA. The reactions were quenched with a final con-
centration of 50 mM EDTA after specific time intervals (2, 4, 6,
and 8 min or 4, 8, 12, and 32 min). The products (dNs) and
substrates (dNTPs) were separated by reversed phase HPLC
using a CAPCELL PAK C18 column (Shiseido Fine Chemicals,
4.6 ϫ 75 mm) and were quantified by peak integration of
absorbance traces at 260 nm, as described previously (27).
Analysis of Kinetic Data—The kinetic data were fitted with
either the Michaelis-Menten equation (Equation 1) or the allo-
steric sigmoidal equation (Equation 2)
Protein Crystallization and X-ray Data Collection—The pro-
tein stock solution for co-crystallization trials contained 5
mg/ml protein, 1 mM MgCl2, 1.8 mM dGTP, 1.8 mM dTTP, 3.6
mM Tris-HCl, and 250 mM NaCl. Crystal screening was done at
room temperature in 96-well Corning crystallization plates.
Protein stock was mixed with the crystallization conditions in
1:1 ratio. Diffraction quality crystals belonging to the orthogo-
v0 ϭ Vmax ϫ ͓dNTP͔/͑Km ϩ ͓dNTP͔͒
(Eq. 1)
v0 ϭ Vmax ϫ ͓dNTP͔h/͑Khalfh ϩ ͓dNTP͔ ͒
(Eq. 2)
h
nal space group P212121 were obtained in 0.1 M buffer contain- where v0 is the initial velocity, Vmax is the maximum velocity,
ing sodium malonate/imidazole/boric acid (at a 2:3:3 molar Km and Khalf are ligand concentrations at half-maximal velocity,
ratio), pH 5.0, and 25% w/v PEG 1500 in a 2-␮l sitting drop at and h is the Hill coefficient.
2816 JOURNAL OF BIOLOGICAL CHEMISTRY
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Allosteric Activation and Inhibition of EF1143 dNTPase
RESULTS
The Overall Structure of EF1143 in Complex with dGTP and
dTTP—The new crystal structure of EF1143 in complex with
dGTP and dTTP (PDB ID 4LRL) demonstrates the same tetra-
meric architecture as the previously reported EF1143 in com-
plex with dGTP and dATP (PDB ID 3IRH, root mean square
deviation between the two structures is 0.67 Å over 1631 C␣)
and, likely, represents the biological assembly of EF1143 in
solution (23). Each EF1143 monomer consists of a bowl-shaped
catalytic HD domain (residues 1–329) and a C-terminal sub-
domain (residues 330–456), shown with pale tones in Fig. 1A.
Four dGTP molecules in the 4LRL structure were found at the
previously identified allosteric sites in the interfaces between
the monomers. The detailed description of these dGTP-bind-
ing sites can be found elsewhere (23) and is reiterated in Fig. 1B.
Guanine base forms five hydrogen-bonding interactions with
the side chains of Asn-36, Gln-41, and Arg-44, providing base
type specificity. The specificity for dGTP binding at this site was
confirmed by kinetic studies, as described in a later section (see
Fig. 5). Surprisingly, however, in the 4LRL structure, the elec-
tron density of dTTP was observed next to dGTP, indicating the
existence of a secondary allosteric site (supplemental Fig. S1).
This contrasts with the 3IRH structure, where dATP was found
in the catalytic site.
There are four identical secondary allosteric sites per
tetramer, but only two were occupied by dTTP in 4LRL. Several
distinct features typify the site (Fig. 1C). First, residues belong-
ing to three different chains (particularly, B, C, and D) form the
binding pocket. Second, the site appears to be specific for the
deoxyribose moiety, with the 3Ј-hydroxyl group interacting
with the main chain atoms of Thr-55 (chain C) and Arg-17
(chain D). The substitution of the deoxyribose with the ribose
moiety would result in structural clash between the 2Ј-hydroxyl
group and Phe-56 (chain C). Third, the triphosphate moiety,
specifically, oxygen atoms of the P␣ and P groups, is recog-
nized by His-245 of chain C and Arg-209 o␤f chain B. Interest-
ingly, one of the P␣ oxygen atoms also has an H-bond with the
deoxyribose hydroxyl group of dGTP. Fourth, the guanidinium
group of Arg-17 in chain D interacts with the ortho-carbonyl
group of thymine. The fact that only one of the three functional
groups of the dTTP base is involved in H-bonding suggests the
promiscuous nature of the secondary site. This is in stark con-
trast to the specific dGTP binding mode at the primary allos-
teric site. The promiscuity of this site may play a key role in
regulation of the hydrolase activity, depending on the presence
of particular dNTPs as elaborated in later sections.
tones, whereas the C-terminal subdomains are shown in pale shades. Dica-
tion metal atoms in the catalytic centers are shown as black spheres. dGTP
(cyan spheres) occupies each of four primary allosteric sites, and dTTP (red
spheres) occupies two (those of chains A and C) of four secondary regulatory
sites. B, dGTP (cyan carbon atoms) binding at the primary allosteric site in
4LRL. Chains B, C, and D are shown in yellow, green, and orange, respectively.
dTTP is shown in gray stick representation. H-bonds are depicted by dashed
lines, and H-bond lengths are shown. This binding site was also described in
FIGURE 1. Crystal structure of EF1143 in complex with dGTP and dTTP. A, our previous publication (23). C, details of the dTTP (pink carbon atoms) inter-
general view of the EF1143 homo-tetramer bound to dGTP and dTTP (PDB ID: action at the secondary allosteric site of 4LRL. Chains B, C, and D are depicted
4LRL). Each monomer is identified with distinct colors and labeled as chains as in B. A dGTP molecule, in the primary allosteric site, is shown in gray.
A–D. The N-terminal catalytic domain of each subunit is colored in brighter Selected side chains are shown and labeled. H-bond lengths are indicated.
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Allosteric Activation and Inhibition of EF1143 dNTPase
FIGURE 2. Modeling of EF1143 activity regulation by dGTP/dTTP binding at the allosteric sites. A, superposition of chain C (green) from 4LRL (EF1143/
dGTP/dTTP) with chain B (gray) of 3IRH (EF1143/dGTP/dATP) using the H . . . HD . . . D coordination motifs. Chain B (yellow), dGTP (cyan spheres), and dTTP (red
spheres) in 4LRL, as well as dATP (gray/red/blue spheres), are shown. Selected side chains are shown and labeled. Note that chain B of 3IRH, rather than chain C,
wasusedforthesuperpositionbecausechainCdoesnotcontaindATPattheactivesite. B, superpositionofchainC(green)from4LRLwithchainA(gray)of3IRH.
Binding of the dTTP molecule (represented by spheres and sticks) to chain C (4LRL) changes loop 196–202 and, particularly, the configuration of Tyr-199, which
otherwise would clash with dTTP.
dTTP Binding Alters the Geometry of EF1143 near the Active
Site—Superposition of chain B of 3IRH and chain C of 4LRL
(Fig. 2A) reveals a potential structural mechanism for modula-
tion of the dNTPase activity by dTTP binding to the secondary
allosteric site. The dTTP-binding site is separated from the cat-
alytic site by a helix-turn-helix fragment comprising ␣13 . . .
␣14. The phenol plane of residue Tyr-243, in the ␣13 . . . ␣14
loop, is in close proximity to the deoxyribose moiety of dATP.
The position of this residue would regulate the location of
the P␣ group, a nucleophilic attack target of the hydroxide
ion stabilized by His-114 (supplemental Fig. S2). The dis-
tance between C␥ of Tyr-243 and C1 of the dATP ribose in
3IRH is 3.72 Å, on average, whereas the same positions are
3.57 Å apart, on average, in the substrate-free forms when
dATP is modeled in the structure. This suggests, first, that
the ␣13 . . . ␣14 helix-turn-helix fragment is mobile and, sec-
FIGURE 3. Mutational analysis of EF1143 validates the structure. Wild-
ond, that dATP binding tends to expand the binding site. In
type (WT) or mutant EF1143 (0.1 ␮M) was incubated in a reaction mixture
containing 0.8 ␮M dGTP and 1 mM dATP. The rate of dATP conversion to dA
was calculated for each mutant and is presented as the percentage of activity
relative to WT. Error bars, S.D.
contrast to the dATP effect, dTTP binding would change the
C␥ . . . C1 distance to an average of 3.32 Å. This 0.40 Å short-
ening could be directly linked to the dTTP steric contact
with residue Gln-241 on the opposite side of ␣13, causing
Tyr-243 to shift into the catalytic site. Thus, dTTP binding “spring” that ensures the reversibility of ligand binding to the
results in contraction of the catalytic site.
secondary allosteric site.
An additional structural change related to dTTP binding at
Mutational Analysis Combined with Activity Assays Corre-
the second allosteric site is the movement of Tyr-199 and the lates with the Structural Features—Our initial biochemical
loop that includes residues 195–202 (Fig. 2B). In the absence of analysis demonstrated that, in the presence of Mg2ϩ, the hydro-
ligand, Tyr-199 protrudes into the allosteric site. Thus, in the lytic activity of EF1143 depended on the presence of at least two
dTTP-bound state, the 195–202 loop must change its confor- different dNTPs, one of which had to be dGTP (23). In this
mation, resulting in an almost 6.9 Å movement of the Tyr-199 study, we measured the dATPase activity of EF1143 in the pres-
C␥ position out of the secondary allosteric site. This new loop ence of both dATP and dGTP, with a dATP:dGTP ratio of
conformation is acceptable but more energetically strenuous, 1250:1. To confirm the importance of several critical residues
as suggested by the Ramachandran plot distribution; in the revealed from the 3IRH and 4LRL structures, several mutants
dTTP-bound state, three residues move from the preferred into were generated and tested for dATPase activity (Fig. 3). First,
the allowed regions (data not shown). This observation suggests analysis of 3IRH, with dATP at the active site, suggested that the
that Tyr-199 and the associated loop could play the role of a catalytic residues comprise a Glu-122-His-129 dyad plus His-
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Allosteric Activation and Inhibition of EF1143 dNTPase
A
B 8
8
6
4
2
0
6
4
2
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
C 8
D
8
6
4
2
0
6
4
2
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
FIGURE 4. dNTPase activities of EF1143 in various mixtures of dNTPs. EF1143 (0.1 ␮M) was incubated with various combinations of dNTP (100 ␮M
each), as indicated on the abscissa. A–D, the rates of dGTP conversion to dG (A), dATP to dA (B), dCTP to dC (C), and dTTP to dT (D) in each condition are
shown on the ordinate. The individual rates were normalized to the dT conversion rate in the presence of all four dNTP (column 8 in D). Error bars, S.D.
arb. unit, arbitrary unit.
114, which together facilitate the in-line nucleophilic attack of a
Survey of dNTPase Activity with Combinations of dNTPs—
hydrolytic water molecule at the ␣-phosphate center of the Our discovery of the promiscuous second allosteric site in 4LRL
dNTP substrate (supplemental Fig. S2). Negligible catalytic prompted us to speculate that the enzymatic activity of EF1143 is
activity of the H114A and H129A mutants supports this differentially regulated, depending on which dNTP occupies this
hypothesis (Fig. 3). Second, dGTP binding at the primary allo- regulatory site. To gain more insight into this, we first measured
steric site features two distinctive patterns (Fig. 1B): ionic inter- the dNTPase activity of EF1143 against each dNTP, in the absence
actions between the triphosphate and basic residues (Lys-14, or presence of one, two, or three additional dNTPs (Fig. 4). Con-
Arg-326, and Lys-330) and hydrogen bonding between the gua- sistentwithourpreviousstudy(23), EF1143failedtohydrolyzeany
nine base and hydrophilic residues (Asn-36, Gln-41 and Arg- of the dNTPs when a sole dNTP was present in the reaction mix-
44). All these residues (except Arg-44) were mutated, individ- ture (Fig. 4, A–D, column 1 in each). Next, the data confirmed our
ually, to Ala, and their dATPase activities were compared. With previous results that, among the four dNTPs, only dGTP could
the exception of R326A, all the mutants were significantly function as an activator of dATP and dCTP hydrolysis (Fig. 4, B
defective in dNTP hydrolysis. Finally, residues observed to and C, columns 2–4). When the rate of dGTP hydrolysis was com-
interact with dTTP in 4LRL, at the second allosteric site, were pared among reaction mixtures containing two dNTP types,
mutated. Although mutation at Arg-17, Tyr-199, or Gln-241 dGTP hydrolysis was clearly enhanced by the presence of dATP or
resulted in significant loss of dATPase activity, R206Q or R209S dCTP, but not dTTP, with dATP being the more potent activator
showed only a moderate decrease in hydrolysis. However, dou- (Fig. 4A, columns 2–4). Further, dTTP hydrolysis was virtually
ble mutation of Arg-206 and Arg-209 resulted in complete loss nonexistent when any one other dNTP was combined with dTTP
of the EF1143 catalytic activity. Taken together, this mutagen- in the reaction mixture (Fig. 4D, columns 2–4). This observation is
esis analysis confirms the importance of residues observed to be consistentwith4LRL, wheredTTPbindingatthesecondallosteric
involved in ligand binding at the catalytic site and the two allo- site shrinks the catalytic site. However, dTTP hydrolysis did occur
steric sites.
when both dGTP and dATP were present (Fig. 4D, column 5),
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Allosteric Activation and Inhibition of EF1143 dNTPase
A
B
dA
dC
+ dATP 1mM
+ dCTP 1mM
100
80
60
40
20
0
150
100
50
0
0
50
100
150
200
250
0.0
0.2
0.4
0.6
0.8
1.0
dGTP (µM)
dGTP (µM)
C
D
with dATP
with dCTP
400
300
200
100
0
20
15
10
5
dC
dA
0
0
200
400
600
800
0
200
400
600
dATP or dCTP (µM)
dATP or dCTP (µM)
FIGURE5. EnzymekineticsofdGTP-, dATP-, anddCTP-dependentdNTPaseactivityofEF1143. A, therateofdATP(circles)ordCTP(squares)conversionwas
determined in the presence of dGTP (0–0.8 ␮M), with an initial dATP or dCTP concentration of 1 mM. The data were fitted with the Michaelis-Menten model
(Equation 1). Vmax and Km were determined to be 47.0 Ϯ 3.4 nmol/nmolꢁmin and 0.17 Ϯ 0.05 ␮M, respectively, for dATP, and 11.5 Ϯ 1.7 nmol/nmolꢁmin and
0.17 Ϯ 0.08 ␮M, respectively, for dCTP. B, the rate of dGTP conversion was determined with the initial dGTP concentration ranging from 0 to 200 ␮M and a fixed
concentration (1 mM) of dATP (circles) or dCTP (squares). Vmax and Km were determined to be 47.3 Ϯ 17.8 nmol/nmolꢁmin and 315 Ϯ 170 ␮M, respectively, for
the dGTP/dATP mixture, and 25.9 Ϯ 7.3 nmol/nmolꢁmin and 213 Ϯ 98 ␮M, respectively, for the dGTP/dCTP mixture when the data were fitted with the
Michaelis-Menten model (Equation 1). C, the rate of dATP (circles) or dCTP (squares) conversion was determined in the presence of increasing concentrations
ofdATP(circles)ordCTP(squares)andafixedconcentrationofdGTP(16␮M). Thedatawerefittedwithanallostericsigmoidalmodel(Equation2). Vmax, Khalf, and
Hill coefficient (h) were determined to be 54.1 Ϯ 7.0 nmol/nmolꢁmin, 310 Ϯ 63 ␮M, and 1.5 Ϯ 0.3, respectively, for dATP. The kinetic values for dCTP were 86.2 Ϯ
7.8 nmol/nmolꢁmin, 376 Ϯ 36 ␮M, and 2.7 Ϯ 0.5, respectively. D, the rate of dG production was also determined from the experiments performed in C, and the
data were fitted with an allosteric sigmoidal model. Vmax, Khalf, and h for dGTPase activity in the presence of dATP (circles) were determined to be 4.2 Ϯ 0.1
nmol/nmolꢁmin, 35 Ϯ 4 ␮M, and 2.7 Ϯ 0.7, respectively. In the presence of varying dCTP concentrations (squares), the kinetic values for dGTPase activity were
1.8 Ϯ 0.3 nmol/nmolꢁmin, 172 Ϯ 46 ␮M, and 2.4 Ϯ 1.4, respectively. Error bars, S.D.
suggesting that both dATP and dGTP are co-activators for hydro- determined in the presence of increasing concentrations of
lysis of dTTP, with dGTP as the primary activator. In contrast, dGTP (Fig. 5A). Both sets of data were fitted with the Michaelis-
dTTP does not activate or enhance the enzyme’s activity against Menten model (Equation 1), suggesting a simple model of
anydNTP. Forexample, theadditionofdTTPtoreactionmixtures dGTP binding at the primary allosteric site. Interestingly, the
that contained dGTP and dATP or dCTP had no effect on the
Km values were similar for both dATP and dCTP (0.17 Ϯ 0.05
hydrolysis of dATP (Fig. 4B, column 2 versus column 7) or dCTP and 0.17 Ϯ 0.08 ␮M, respectively). This suggests that dGTP
(Fig. 4C, column 2 versus column 6) at the initial concentrations binding at the allosteric site is independent of dATP and dCTP
tested. With all four dNTPs present at equal concentrations, the binding at the catalytic site. Further kinetic analyses, with
order of dNTP hydrolysis was dGTP Ͼ dCTP Ͼ dATP Ͼ dTTP higher dGTP concentrations, suggested that dGTP binding at
(Fig. 4, A–D, compare column 8 from each panel). Based on all the catalytic site also follows the Michaelis-Menten model
these comparisons, we conclude that dGTP is a primary activator (Equation 1) (Fig. 5B). The rates of dATP and dCTP hydrolysis
because appreciable dNTP hydrolysis was not observed in any were moderately constant over the range of dGTP concentra-
reaction mixture in the absence of dGTP. This conclusion is con- tions examined (supplemental Fig. S3). Taken together, the
sistent with the crystal structures of EF1143, with dGTP at the data suggest that dGTP has a strong (ϳ2000-fold) preference
primary allosteric site (Fig. 1). In addition, dATP is a stronger sec- for the allosteric site as compared with the catalytic site.
ondary activator than dCTP because the rates of dGTP and dTTP
hydrolysis were higher with dATP in the reaction mixture as com- further understand the activation features of EF1143 by dATP
pared with those with dCTP. and dCTP (Fig. 4), dNTPase assays were performed with
dATP and dCTP Allosterically Activate the dNTPase—To
Determination of Kinetic Constants of dGTP at the Allosteric increasing concentrations of dATP or dCTP in a reaction mix-
Site and Catalytic Site—Analysis of the two crystal structures ture containing a fixed concentration of dGTP, 16 ␮M, at which
(3IRH and 4LRL), along with our dNTPase activity survey (Fig. the primary allosteric site of the enzyme is fully occupied by
4), suggests that dGTP binding at the primary allosteric site is a dGTP (Kd of dGTP at the primary allosteric site is 0.17 ␮M).
prerequisite for the dATP- and dCTP-dependent activation of When the rates of dA and dC accumulation were plotted as a
EF1143. To determine the binding constant for dGTP at the function of dATP and dCTP concentrations, respectively, the
allosteric site, the rates of dATP and dCTP hydrolysis were data were best fitted with sigmoidal curves (Equation 2) (Fig.
2820 JOURNAL OF BIOLOGICAL CHEMISTRY
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Allosteric Activation and Inhibition of EF1143 dNTPase
A
B
C
with dGTP 16 µM, dATP 1mM
with dGTP 16 µM, dCTP 1mM
40
30
20
10
0
500
10
8
dA with dGTP 16 µM, dATP 1 mM
dC with dGTP 16 µM, dATP 1 mM
with dGTP 16 µM, dATP 1mM
with dGTP 16 µM, dCTP 1mM
400
300
200
100
0
6
4
2
0
0
500
1000
1500
2000
0
500
1000
1500
2000
0
500
1000
1500
2000
dTTP (µM)
dTTP (µM)
dTTP (µM)
D
E
dA with dATP 1mM
dC with dCTP 1mM
500
400
300
200
100
0
with dATP 1mM
with dCTP 1mM
100
80
60
40
20
0
0
200
400
600
800
1000
0
200
400
600
800
1000
dGTP (µM)
dGTP (µM)
FIGURE 6. dTTP and dGTP at high concentrations inhibit dNTPase activity of EF1143. The dNTPase activity of EF1143 was determined in the presence of
increasing concentrations of dTTP (0–1600 ␮M) and fixed concentrations of dGTP (16 ␮M) and dATP (1 mM) or dCTP (1 mM). A–C, the rates of dTTP (A), dGTP (B),
and dATP and dCTP (C) conversion were plotted relative to dTTP concentration. D, the rate of dGTP triphosphohydrolase activity was determined in the
presence of increasing concentrations of dGTP (100–800 ␮M) in the presence of either 1 mM dATP or 1 mM dCTP. E, the rate of dATP (circles) or dCTP (squares)
conversion is plotted for the experiments performed in D. Error bars, S.D.
5C), indicating allostery. The rate of dC accumulation was more dG, dA, and dC conversion were monitored with increasing
sensitive to dCTP concentration than the rate of dA accumula- concentrations of dTTP (Fig. 6, B and C).
tion was to dATP, resulting in a higher Hill coefficient value and
The 4LRL structure provides mechanistic insight into the
V
max for dCTP than dATP. When the rate of dG accumulation inhibition mechanism by dTTP. The ligand interacts with res-
was plotted, dATP was far superior to dCTP in allosteric acti- idue Gln-241 of helix ␣13, shifting Tyr-243 toward the catalytic
vation of dGTP hydrolysis (Fig. 5D). These data support the binding site by ϳ0.4 Å (Fig. 2A). This movement could either
observation that dATP is a more potent activator of dGTP prevent substrate binding or disrupt correct positioning of the
hydrolysis than dCTP, as seen when all dNTPs were present at dNTP for nucleophilic attack. Modeling of dGTP ligand bind-
100 ␮M (Fig. 4A, columns 2 and 3). Further, at 100 ␮M, the rate ing at the second allosteric site (Fig. 7) suggests that it could
of dA accumulation is slightly higher than that of dC accumu- exert a similar effect on helix ␣13/Gln-241, inhibiting enzyme
lation (Fig. 5C), which also agrees with the data presented in Fig. catalysis. Consistent with this notion, at high dGTP concentra-
4. Taken together, the results show that dATP and dCTP allo- tions (greater than 200 ␮M), the dNTPase was inhibited (Fig. 6,
sterically activate the dNTPase with a different cooperativity for D and E). The dCTP molecule should be able to bind to the
substrates. Our structural modeling suggests that both dATP secondary allosteric site in a mode similar to that of dTTP.
and dCTP might bind at the secondary allosteric site, which is However, the deoxycytidine, in particular the amino group in
occupied by dTTP in 4LRL (see the section below).
para-position, would clash with Gln-241 if dCTP binds exactly
dTTP and dGTP Inhibits dNTPase Activity at High in the same conformation as observed for dTTP in 4LRL. A
Concentration—Comparison of crystal structures 3IRH and similar observation could be made for dATP in either anti-
4LRL (Fig. 2) suggests that dTTP binding at the secondary allo- conformation or syn-conformation (Fig. 7). Despite these cave-
steric site could negatively influence EF1143 catalytic activity ats, the binding pocket appears to be large enough to allow
(Fig. 4). To further investigate this possibility, we measured the cytosine/adenine plane rotation, for example, toward the
rate of dT accumulation in increasing concentrations of dTTP Arg-236 residue that would prevent collision with Gln-241.
with a fixed concentration of dGTP and dATP or dCTP (Fig. The reduced, but still significant activity of the Q241A
6A). Note that hydrolysis of dTTP can only be observed in the mutant in the dGTP/dATP mixture (Fig. 3) may be inter-
presence of both dGTP and dATP or dCTP (Fig. 4D). The rate preted to support this hypothesis. Therefore, the steric dif-
of dTTP hydrolysis increased with increasing concentrations of ference exerted by the amino group in dCTP/dATP and the
dTTP, up to 400 ␮M, and then gradually declined at higher carbonyl group in the analogous position of dTTP/dGTP
concentrations. Strikingly, no conversion of dTTP was may differently affect the position of helix ␣13 and residue
observed at 1600 ␮M dTTP with dGTP and dCTP in the reac- Tyr-243 in the catalytic site, thus, switching the enzyme
tion mixture. Similar results were observed when the rates of between the inhibited and the activated states. Taken alto-
JANUARY 31, 2014•VOLUME 289•NUMBER 5
JOURNAL OF BIOLOGICAL CHEMISTRY 2821

Allosteric Activation and Inhibition of EF1143 dNTPase
FIGURE 7. Modeling of dNTP binding at the secondary allosteric site. A, modeling of the dGTP (cyan carbon atoms) and dATP (yellow carbon atoms)
molecules in syn-conformation into the secondary allosteric site, formed by chains B (yellow), C (green), and D (orange) in 4LRL. The dGTP molecule in the
primary allosteric site is shown as gray sticks, whereas the dTTP molecule in the second site is shown as pink/blue/red lines. H-bonding networks for the dTTP
molecule are shown as black dashes, as well as the shortest distance (3.1 Å) between dTTP and Gln-241. In both syn-configuration and anti-configuration (see
panel B), the dGTP molecule carbonyl group positions in the same place as the para-carbonyl in dTTP, whereas in the case of dATP, the bulkier amino group
occupies this space. This suggests that dGTP might bind to the secondary allosteric site and exert the same inhibitory effect as dTTP. In contrast, dATP would,
probably, rotate toward Arg-206 (to avoid the clash with Gln-241), which could weaken its influence on the active site. B, modeling of dGTP and dATP, in
anti-conformation, into the secondary allosteric site. Color-coding is identical to panel A. H-bonding network and shortest distance to Gln-241 for the dTTP
molecule are shown as black dashes within 3.1 Å distance.
dGTP
dTTP
gether, the kinetic data support a modulatory role of the
X
X
secondary allosteric site upon binding to different dNTPs.
DISCUSSION
X
X
The proper balance of cellular dNTP pools is critical for
maintaining genome stability. The central role of ribonucle-
oside diphosphate reductases in this has been well docu-
mented (4). A recent study of SAMHD1, a mammalian
dNTPase, suggested its essential role in balancing dNTP
pools in concert with the ribonucleoside diphosphate reduc-
tase (5). We and others elucidated the structural basis of
dGTP-dependent SAMHD1 tetramerization and allosteric
activation (36, 37). In this study, we present a crystal struc-
ture of EF1143 in complex with dGTP and dTTP (4LRL) and
provide insight into an elegant allosteric mechanism of
dNTP-mediated inhibition of enzyme catalysis.
B
C
dGTP
A
Inhibited form
dCTP
dATP
D
O
O
Intermediate
O
O
Active form
FIGURE 8. Model of dNTP-dependent allosteric regulation of EF1143
catalysis. dGTP binds to each of four primary allosteric sites (Intermediate).
Binding of either dTTP or dGTP at the two secondary allosteric sites, adjacent
to the primary site, results in an inhibited form, by closing the catalytic site (X),
whereas binding of dATP or dCTP at the same secondary sites activates the
enzyme (O).
Comparison of 4LRL with the apo-EF1143 (2O6I) and dGTP/
dATP bound forms (3IRH), along with extensive enzyme kinetic
studies, presents a qualitative model for EF1143 allosteric regula-
tion (Fig. 8). dGTP binding at the primary allosteric site is neces-
sary for hydrolysis of the canonical dNTP substrates. Analysis of bacteriophage T4 infection (38–40). At these concentrations,
the N36A and Q41A mutants confirmed the critical impor- the primary allosteric site should be fully occupied with dGTP
tance of the guanine base-recognizing residues (Fig. 3). The and substrate hydrolysis at the catalytic site could be modulated
enzyme kinetic analysis indicated high specificity of the pri- by different dNTPs binding at the secondary allosteric sites.
mary allosteric site for dGTP (Fig. 5A). Taken together, binding
To assess whether the proposed mechanism of EF1143 reg-
of dGTP could be considered an enzyme preactivation step. ulation by dNTPs could serve as a general model for homo-
Then, the enzyme can be further activated or inhibited by tetrameric dNTPases, we compared the amino acid sequences
dNTPs binding to the secondary allosteric site, with different of EF1143 with 43 of its closest homologs, mostly from Entero-
affinities. If the site is occupied by dATP or dCTP, the enzyme coccus and Lactobacillus species (at least 97% coverage and 65%
becomes catalytically active (Fig. 5, C and D). With dGTP or identity, supplemental Fig. S4). Among the closest sequence
dTTP bound at the site, EF1143 is negatively regulated (Fig. 6). homologs, most of the critical residues in the catalytic and two
The physiological concentrations of dNTPs in prokaryotes are allosteric sites are 100% conserved (supplemental Fig. S4, red
in the range of 100–300 ␮M and fluctuate several folds during boxes). We also carried out a structure-assisted comparison of
2822 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 5•JANUARY 31, 2014

Allosteric Activation and Inhibition of EF1143 dNTPase
Mutat. Res. 318, 1–64
EF1143 and SAMHD1 (supplemental Fig. S5). The human
SAMHD1 dNTPase (sequence identity 36% over 274 residues)
is a structural homolog of EF1143. For the conservation analy-
sis, we superimposed SAMHD1 chain A from 4BZB (dGTP-
bound tetrameric form) to chain C of EF1143 tetramer (supple-
mental Fig. S5A). Overall, all the dNTP-binding sites between
EF1143 and SAMHD1 are highly conserved. At the dGTP-spe-
cific primary allosteric site, two critical residues for EF1143
preactivation, Arg-44 and Arg-326, have Arg-145 and Arg-451
as their counterparts in SAMHD1 (supplemental Fig. S5B). Of
note, SAMHD1 R145A mutation resulted in less than 1% of
wild-type dNTPase activity (6). Interestingly, hydrogen-bond-
ing interactions between the base of deoxyribonucleoside
triphosphate and surrounding residues in both enzymes sug-
gest a promiscuity of the secondary allosteric site to other
dNTPs (Fig. 1C) (36, 37). The catalytic residues Glu-122-His-
129-His-114 in EF1143 correspond to Asp-218-His-233-His-
210 in SAMHD1 (supplemental Fig. S5C). Thus, the hydrolysis
in both enzymes may proceed in a similar manner, through the
nucleophilic attack of the activated water species (hydroxide
ion) on the P␣ center.
3. Kumar, D., Abdulovic, A. L., Viberg, J., Nilsson, A. K., Kunkel, T. A., and
Chabes, A. (2011) Mechanisms of mutagenesis in vivo due to imbalanced
dNTP pools. Nucleic Acids Res. 39, 1360–1371
4. Nordlund, P., and Reichard, P. (2006) Ribonucleotide reductases. Annu.
Rev. Biochem. 75, 681–706
5. Franzolin, E., Pontarin, G., Rampazzo, C., Miazzi, C., Ferraro, P., Palumbo,
E., Reichard, P., and Bianchi, V. (2013) The deoxynucleotide triphospho-
hydrolase SAMHD1 is a major regulator of DNA precursor pools in mam-
malian cells. Proc. Natl. Acad. Sci. U.S.A. 110, 14272–14277
6. Goldstone, D. C., Ennis-Adeniran, V., Hedden, J. J., Groom, H. C., Rice,
G. I., Christodoulou, E., Walker, P. A., Kelly, G., Haire, L. F., Yap, M. W., de
Carvalho, L. P., Stoye, J. P., Crow, Y. J., Taylor, I. A., and Webb, M. (2011)
HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate
triphosphohydrolase. Nature 480, 379–382
7. Powell, R. D., Holland, P. J., Hollis, T., and Perrino, F. W. (2011) Aicardi-
Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a
dGTP-regulated deoxynucleotide triphosphohydrolase. J. Biol. Chem.
286, 43596–43600
8. Berger, A., Sommer, A. F., Zwarg, J., Hamdorf, M., Welzel, K., Esly, N.,
Panitz, S., Reuter, A., Ramos, I., Jatiani, A., Mulder, L. C., Fernandez-
Sesma, A., Rutsch, F., Simon, V., Ko¨nig, R., and Flory, E. (2011) SAMHD1-
ϩ
deficient CD14 cells from individuals with Aicardi-Goutieres syndrome
are highly susceptible to HIV-1 infection. PLoS Pathog. 7, e1002425
9. Hrecka, K., Hao, C., Gierszewska, M., Swanson, S. K., Kesik-Brodacka, M.,
Srivastava, S., Florens, L., Washburn, M. P., and Skowronski, J. (2011) Vpx
relieves inhibition of HIV-1 infection of macrophages mediated by the
SAMHD1 protein. Nature 474, 658–661
Despite the structural similarities between EF1143 and
SAMHD1, several important differences exist. Although both
apo-bound and dGTP-bound EF1143 are tetramers in solution
(23), SAMHD1 undergoes tetramerization upon dGTP binding
(26, 27). Further, different dNTP binding at the secondary allo-
steric site of EF1143 can result in either active or inhibited forms,
whereas no such regulation has been reported for SAMHD1.
A complete understanding of EF1143 regulation by dNTPs
requires further work. Although our hypothesis that the switch
between the active and inhibited modes could be regulated by
binding of different ligands to the promiscuous secondary allo-
steric site agrees with the biochemical data, our suggestion is
primarily based on modeling into the rigid protein structure.
To prove that dGTP, dATP, and dCTP indeed bind to the sec-
ondary allosteric site, more crystallization experiments with
different dNTP mixtures are needed. The issue also could be
approached theoretically, using flexible molecular docking,
molecular dynamics, or Monte-Carlo simulations, using our
structures as a starting point. Furthermore, in 3IRH, only two
out of four catalytic sites were occupied by dATP, and only two
out of four secondary regulatory sites in 4LRL have dTTP. This
might be an artifact of crystal packing but could also suggest
communication between monomers in the tetrameric assem-
bly. Finally, the function of the prominent and highly conserved
C terminus of EF1143 remains unknown. These three struc-
tural aspects are currently under investigation.
10. Laguette, N., Sobhian, B., Casartelli, N., Ringeard, M., Chable-Bessia, C.,
Se´ge´ral, E., Yatim, A., Emiliani, S., Schwartz, O., and Benkirane, M. (2011)
SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction
factor counteracted by Vpx. Nature 474, 654–657
11. Baldauf, H. M., Pan, X., Erikson, E., Schmidt, S., Daddacha, W., Burggraf,
M., Schenkova, K., Ambiel, I., Wabnitz, G., Gramberg, T., Panitz, S., Flory,
E., Landau, N. R., Sertel, S., Rutsch, F., Lasitschka, F., Kim, B., Ko¨nig, R.,
Fackler, O. T., and Keppler, O. T. (2012) SAMHD1 restricts HIV-1 infec-
ϩ
tion in resting CD4 T cells. Nat. Med. 18, 1682–1687
12. Descours, B., Cribier, A., Chable-Bessia, C., Ayinde, D., Rice, G., Crow, Y.,
Yatim, A., Schwartz, O., Laguette, N., and Benkirane, M. (2012) SAMHD1
ϩ
restricts HIV-1 reverse transcription in quiescent CD4 T-cells. Retrovi-
rology 9, 87
13. St Gelais, C., de Silva, S., Amie, S. M., Coleman, C. M., Hoy, H., Hollen-
baugh, J. A., Kim, B., and Wu, L. (2012) SAMHD1 restricts HIV-1 infection
in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and
ϩ
primary CD4 T-lymphocytes cannot be upregulated by interferons. Ret-
rovirology 9, 105
14. Ahn, J., Hao, C., Yan, J., DeLucia, M., Mehrens, J., Wang, C., Gronenborn,
A. M., and Skowronski, J. (2012) HIV/simian immunodeficiency virus
(SIV) accessory virulence factor Vpx loads the host cell restriction factor
SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1. J. Biol.
Chem. 287, 12550–12558
15. Nakai, H., and Richardson, C. C. (1990) The gene 1.2 protein of bacterio-
phage T7 interacts with the Escherichia coli dGTP triphosphohydrolase to
form a GTP-binding protein. J. Biol. Chem. 265, 4411–4419
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the gene for dGTP triphosphohydrolase from Escherichia coli. Proc. Natl.
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Acknowledgment—We thank Dr. Teresa Brosenitsch for careful read-
ing of the manuscript and editorial help.
17. Wisplinghoff, H., Seifert, H., Wenzel, R. P., and Edmond, M. B. (2003)
Current trends in the epidemiology of nosocomial bloodstream infections
in patients with hematological malignancies and solid neoplasms in hos-
pitals in the United States. Clin. Infect. Dis. 36, 1103–1110
18. National Nosocomial Infections Surveillance System (2004) National
Nosocomial Infections Surveillance (NNIS) System Report, data summary
from January 1992 through June 2004, issued October 2004. Am. J. Infect
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2824 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 5•JANUARY 31, 2014

Enzymology:
Mechanisms of Allosteric Activation and
Inhibition of the Deoxyribonucleoside
Triphosphate Triphosphohydrolase from
Enterococcus faecalis
Ivan I. Vorontsov, Ying Wu, Maria DeLucia,
George Minasov, Jennifer Mehrens, Ludmilla
Shuvalova, Wayne F. Anderson and Jinwoo
Ahn
J. Biol. Chem. 2014, 289:2815-2824.
doi: 10.1074/jbc.M113.524207 originally published online December 12, 2013
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