(Received for publication, October 7, 1994)
From the
An Escherichia coli open reading frame containing
significant homology to the active site of the MutT enzyme codes for a
novel dinucleotide pyrophosphatase. The motif shared by these two
proteins and several others is conserved throughout nature and may
designate a nucleotide-binding or pyrophosphatase domain. The E.
coli NADH pyrophosphatase has been cloned, overexpressed, and
purified to near homogeneity. The protein contains 257 amino acids (M = 29,774) and migrates on gel filtration
columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a
broad range of dinucleotide pyrophosphates, but uniquely prefers the
reduced form of NADH. The V
/K
for NADH (69 µmol min
mg
mM
) is an order of magnitude higher
than for any other dinucleotide pyrophosphate tested. In addition, the K
for NADH (0.1 mM) is 50-fold
lower than the K
for
NAD
. The hydrolysis of dinucleotide pyrophosphates
requires divalent metal ions and yields two mononucleoside
5`-phosphates. The metals that most efficiently stimulate activity are
Mg
and Mn
. Although these metals
support similar V
values at optimal metal
concentration, the apparent K
for
Mg
is 3.7 mM (at 1 mM NADH),
whereas the apparent K
for Mn
at the same NADH concentration is 30 µM.
This work was initiated during a study of a highly conserved
motif that was first identified in the Escherichia coli MutT
and Streptococcus pneumoniae MutX antimutator proteins by
Mejean et al.(1) . The motif is shared by enzymes from Proteus vulgaris(2) , human cells(3) , and
another partially purified E. coli protein(4) .
Because these proteins all hydrolyze nucleoside triphosphates to form
nucleoside monophosphates and inorganic pyrophosphate, we proposed that
this small region of conserved amino acids, common to these proteins,
designates a nucleoside-triphosphate pyrophosphohydrolase
activity(4) . Several other open reading frames in widely
divergent organisms that specify this small sequence of amino acids but
that otherwise have little homology were identified by a computer
search of the data bases(1, 5) . We have initiated a
systematic study to isolate and examine whether these other genes code
for proteins that share similar enzymatic activities. One of these
proteins, coded for by open reading frame orf257 (or yjad) between the thiC and hemeE genes in
the 90-min region of the E. coli genetic map (6) (GenBank accession numbers U00006 and D12624),
is the subject of this study. It will be demonstrated that this protein
does not hydrolyze nucleoside triphosphates, but that it has a novel
NADH pyrophosphatase activity, which provides further insight into the
function of the conserved amino acid motif.
The reaction mixtures (100 µl for Assays 1 and 2 and 50
µl for Assay 3) contained 50 mM Tris-Cl, pH 8.5, 20 mM MgCl, and 0.33-3.3 milliunits of enzyme. One
unit of activity is 1 µmol of NADH hydrolyzed per min. After
incubation for 10-30 min at 37 °C, the reactions were
terminated by boiling. Stage I was then treated as follows.
Figure 1: Alignment of E. coli MutT and E. coli dinucleotide pyrophosphatase amino acid sequences. The amino acid sequences of the MutT and Orf257 proteins are aligned based on the conserved region shown in boldface. Identical amino acids are indicated with connectinglines, and similar amino acids (i.e. hydrophobic, polar, or charged) are noted with colons.
Figure 2:
Expression and purification of the Orf257
protein. A, a 15% SDS-polyacrylamide gel stained with
Coomassie Blue. Lane1 contains 4 µg each of
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
(29 kDa), and MutT (15 kDa). Lanes2 and 3 show the expression of the Orf257 protein. HMS174(DE3) cells
containing the plasmid pETorf257 were grown in LB/ampicillin media as
described under ``Methods.'' Cells (1.5 ml) were harvested
before (lane2) and 2 h after (lane3) isopropyl--D-thiogalactopyranoside
addition. The A
/ml was determined for each
aliquot, and the cells were boiled in 400 µl of SDS loading
buffer/A
unit. Lane2 contains
20 µl of the preinduced cell extract. Lane3 contains 20 µl of the induced cell extract. Lanes4 and 5 contain 2 and 6 µg of the purified
protein (Fraction V), respectively. B, a 15% native
polyacrylamide gel stained with Coomassie Blue. Lane1 contains the same standards as lane1 in A. Lanes2 and 3 contains 2 and 6
µg of Fraction V, respectively.
Figure 3:
Products of the E. coli dinucleotide pyrophosphatase. Fraction V (3.0 milliunits) was
incubated with 1 mM NADH, 20 mM MgCl, and
50 mM Tris-Cl, pH 8.5, at 37 °C. The reaction was
terminated by boiling, and aliquots were used for the determination of
AMP (
), inorganic orthophosphate (
), and phosphatase-labile
phosphate (
) as described under
``Methods.''
The pH optimum for the
reaction was 8.5 (data not shown). Both MutT (4) and the
MutT-like enzyme Orf17 ()also have alkaline pH optima.
Although phosphate buffer inhibited the reaction slightly (20%),
dithiothreitol, DNA, monovalent cations, pyrophosphate, and EDTA (at
concentrations significantly lower than metal concentration) had no
effect on activity. The lack of inhibition or stimulation by
pyrophosphate clearly distinguished the enzyme from the reversible
NAD
pyrophosphorylase (EC 2.7.7.1)(17) . Only
weak product inhibition was observed for NMNH, which indicates that the
reaction is not freely reversible. A double reciprocal analysis of four
titrations with NADH (0.2-1.0 mM) in the presence of
various concentrations of NMNH (0-2.1 mM) suggested that
NMNH is a linear competitive inhibitor, with a K
of 4.2 mM. No inhibition was observed for the oxidized
NMN
even at 10-fold higher inhibitor concentrations
(data not shown).
The enzyme was
more active on NADH than on any other substrate tested, and in each
case involving the pyridine nucleotides, the reduced substrate was
hydrolyzed more rapidly than its respective oxidized form. Several
other nucleotides with related structural features showed varying
degrees of reactivity. However, it is especially noteworthy that none
of the 16 canonical (deoxy)nucleoside di- and triphosphates were
hydrolyzed. This is in striking contrast to the other enzymes of the
family (MutT, MutX, Orf17, and human 8-oxo-dGTPase), which all show a
marked preference for deoxynucleoside triphosphates. Certain patterns
emerge in comparing the reactivity of several of the other substrates.
For example, the rate of hydrolysis decreases as the length of the
polyphosphate bridge increases in the ApA series.
Furthermore, a second nucleotide is essential for optimal activity. The
elimination of the nicotinamide group (ADP-ribose) causes a 3-fold
reduction in rate, and the replacement of the ribose with a glucose
(ADP-glucose) causes a further 7-fold decrease. The enzyme has no
detectable activity under these conditions toward UDP-glucose in which
neither the adenine nor the nicotinamide groups are retained. These
observations focus attention on structural features of the substrates
that correlate with their reactivity. However, caution should be
exercised in overinterpreting the observations made on this small group
of compounds examined under one set of reaction conditions.
Several
of the substrates showing high relative rates (in Table 1) were
subjected to a more detailed kinetic analysis. Double reciprocal
(Lineweaver-Burk) analysis (18) and nonlinear least-squares
fitting weighted to substrate concentration (19) were used to
determine the K and V
values of the substrates tested. Table 2lists the K
, V
, and catalytic
efficiency (V
/K
) of various
substrates, which again demonstrate that NADH is the most favored
substrate for the Orf257 protein identified so far. The replacement of
the adenine moiety with hypoxanthine affects the K
or V
slightly (<3-fold). However,
changes on the nicotinamide nucleotide have much larger effects on both
the K
and V
. The
substitution of the nicotinamide group with adenine (AppA) or the
elimination of the base entirely (ADP-ribose) causes an order of
magnitude decrease in V
/K
,
and the introduction of a positive charge on the nicotinamide is
associated with a decrease in V
/K
by over 2 orders of magnitude.
The striking preference for
NADH over NAD, which may be related to the
physiological function of the Orf257 protein, is clearly demonstrated
in Fig. 4. For NADH, the V
(7.6 units
mg
) corresponds to a turnover rate of 3.8/s based on
two active sites/dimer. This is similar to the k
for the MutT enzyme(14, 20) .
Figure 4:
Preference of the E. coli dinucleotide pyrophosphatase for the reduced form of NAD. A, initial velocity versus substrate concentration
for NADH () and NAD
(
). Reactions (50
µl) contained 20 mM MgCl
, 50 mM Tris-Cl, pH 8.5, and 0.5 milliunit of enzyme (Fraction V) for NADH
or 3.7 milliunits of enzyme (Fraction V) for NAD
.
Reactions were terminated by boiling and analyzed by Assay 3 (see
``Methods''). Curves are fit using nonlinear least-squares
analyses(19) . B, double reciprocal plot of the data
in A.
Fig. 5shows a titration of 1 mM NADH with
MnCl or MgCl
. Double reciprocal analysis (Fig. 5B) yields an apparent V
for the Mn
-activated reaction of 22 units/mg
with an apparent K
(Mn
) of 30
µM and an apparent V
for the
Mg
-activated reaction of 8.8 units/mg with an
apparent K
(Mg
) of 3.7
mM. This indicates that at low metal concentrations,
Mn
has a 310-fold higher V
/K
(metal) than
Mg
. However, due to the inhibitory effects of
Mn
, the two metals support roughly equal velocities
at high metal concentrations.
Figure 5:
Activation of the E. coli dinucleotide pyrophosphatase by divalent metal cations. A, initial velocity versus concentration of
MgCl (
) and MnCl
(
). Reactions (100
µl) were run at 37 °C with 1 mM NADH, 50 mM Tris-Cl, pH 7.5, with 0.73 milliunit of enzyme (Fraction V).
Reactions were terminated by boiling, and AMP was measured in each
using Assay 2. B, double reciprocal plot of the data in A, fit by least-squares analysis weighted to substrate
concentration(19) , excluding points above 1 mM MnCl
.
Since the purification and characterization of NAD pyrophosphatase (EC 3.6.1.22) from potato extracts were described
by Kornberg and Pricer(22) , enzymes cleaving NAD
into NMN
and AMP have been reported in a large
variety of plants, animals, and bacteria. The relatedness of these
enzymes has been difficult to ascertain because few have been purified,
and most have a broad spectrum of activities not only to NAD
and NADP
, but also to FAD and ADP-ribose and, in
some cases, to ATP, ADP, and the family of bis(5`-nucleosidyl)
oligophosphates as represented by, for example, diadenosine
tetraphosphate (AppppA). However, only one other enzymatic activity has
been reported that cleaves NADH at a higher rate than
NAD
, as is the case with the enzyme described in this
paper. Jacobson and Kaplan (23) partially purified a
dinucleotide pyrophosphatase from pigeon liver extracts that hydrolyzed
NADH at a higher rate than NAD
, but their enzyme is
clearly distinguished from the E. coli enzyme by several
criteria including its activity on
-NAD
and its
preference for ADP-ribose, which it hydrolyzes at 200% of the rate of
NADH. The E. coli enzyme is not active on
-NAD
, and its V
/K
for NADH is 20-fold
higher than for ADP-ribose. Further comparisons of the E. coli enzyme described in this paper with previously reported related
activities is complicated by the fact that the earlier work was done
prior to the development of techniques for gene analysis.
Of what
use to the cell is an enzyme that hydrolyzes and essentially
inactivates an important cofactor in cellular metabolism? One
possibility is that NMNH plays some undiscovered role in the cell. The
only way known, so far, of generating NMNH is through the hydrolysis of
NADH by this enzyme. A second, more general role of the enzyme could be
the regulation of the intracellular NADH/NAD ratio,
which is known to be an important factor in maintaining a balance
between anabolic and catabolic pathways in higher organisms.
Oxidoreduction reactions in which NAD
and NADH
participate as cofactors are, in most cases, freely reversible, and the
selective removal of NADH would favor the oxidative pathway especially
under anaerobic conditions, where the reoxidation of NADH would be
diminished. It is interesting that Jacobson and Kaplan(23) , in
their early work, reconstructed a system in which the addition of
partially purified pigeon liver NADH pyrophosphatase increased the rate
and the extent of formation of acetaldehyde from ethanol by purified
alcohol dehydrogenase. Their interpretation was that the selective
hydrolysis of NADH shifted the equilibrium in favor of the oxidized
substrate. Thus, the NADH pyrophosphatase could substitute for an
electron acceptor and influence the equilibrium of a metabolic pathway
under a specific set of circumstances. We are constructing a null
mutant devoid of the enzyme in order to uncover its functional
significance. Clinical interest has been generated by the report (24, 25) that Lowe's syndrome, a genetic disease
with pleiotropic sequelae, is tied to an overproduction of a
dinucleotide pyrophosphatase.
Our major interest in the enzyme is
that it shares a signature sequence,
GXU(X)ET(X)
REUXEE
(where U represents the bulky aliphatic residue L, I, or V), with four
other proteins that we have partially characterized and that all have
nucleoside-triphosphate hydrolase activities producing nucleoside
monophosphates and inorganic
pyrophosphate(1, 4, 15) . Sakumi et al.(3) have described an 8-oxo-dGTPase activity from human
cells that also has the same amino acid motif. Besides this small
region of identity, the six proteins (including the enzyme described in
this paper) share very little similarity. Our analyses of site-directed
mutations in MutT implicate this small region, common to all the
proteins, as being important in the enzymatic activity of the enzymes (1, 26) , and NMR structural analysis of MutT has
revealed that this region of the protein is involved in nucleotide
binding(27) . Four of the six proteins from E. coli, S. pneumoniae, P. vulgaris, and human cells (1, 2, 3, 15) prevent mutations
caused by AT
CG transversions. A fifth protein, coded for by E. coli orf17, has not, as yet, been shown to prevent
mutations, but it is a nucleoside triphosphatase with a preference for
dATP(4) . These five proteins, which only have this small
signature sequence in common, catalyze the hydrolysis of nucleoside
triphosphates according to the following scheme: (deoxy)nucleoside
triphosphate
(deoxy)nucleoside monophosphate +
PP
. Until we discovered the NADH pyrophosphatase described
in this report, we believed that the signature sequence designated a
catalytic site specific for the attack on the beta-phosphate
of a nucleoside triphosphate with the elimination of
pyrophosphate(28) . Because NADH pyrophosphatase catalyzes the
cleavage of NADH
NMNH + AMP, we believe that this signature
sequence, which in the MutT protein forms a loop-helix-loop motif (27) not seen in other nucleotide-binding sites(29) ,
has been conserved during evolution and adapted to participate in
different metabolic reactions involving the cleavage of a nucleotide
pyrophosphate bond. Computer searches of the sequence data banks (1, 5) have revealed several other genes of unknown
function specifying the same amino acid motif in a variety of organisms
ranging from viruses to eucaryotes. We are in the process of cloning,
expressing, and identifying enzyme activities associated with these
genes. We are also determining the three-dimensional solution structure (27) and crystal structure
of some of these
enzymes. These approaches should lead to an understanding of the basic
biochemistry associated with the conserved amino acid motif and perhaps
identify functions for the other genes containing sequences that
specify this arrangement of amino acids.