(Received for publication, July 19, 1995)
From the
The product of the Escherichia coli orf1.9, or yefc, gene (GenBank accession number L11721)
has been expressed under the control of a T7 promoter, purified to
apparent homogeneity, and identified as a novel enzyme that hydrolyzes
GDP-mannose or GDP-glucose to GDP and the respective hexose. The enzyme
has little or no activity on other nucleotides, dinucleotides,
nucleotide sugars, or sugar phosphates. It has a pH optimum between 9.0
and 9.5, a K
of 0.3 mM, and a V
of 1.6 µmol min
mg
for GDP-mannose, and it requires divalent
cations for activity. This enzyme of 160 amino acids (M
= 18, 405) contains the consensus
sequence
GX(I/L/V)(E/Q)(X)
ET(X)
R(X)
E(X)
(I/L),
characteristic of the MutT family of proteins and previously shown to
form part of the nucleotide-binding site of MutT (Frick, D. N., Weber,
D. J., Abeygunawardana, C., Gittis, A. G., Bessman, M. J., and Mildvan,
A. S.(1995) Biochemistry 34, 5577-5586). A comparison of
the enzymatic reactions catalyzed by the GDP-mannose mannosyl hydrolase
and the other enzymes of the MutT family suggests that the consensus
signature sequence designates a novel nucleoside diphosphate binding
site and catalytic motif.
Many primary amino acid ``signature'' sequences have
been identified that designate protein sites involved in ligand binding
or enzymatic catalysis. One of these signature sequences was discovered
as part of the Escherichia coli MutT and Streptococcus
pneumoniae MutX antimutator proteins (1, 2) and
independently by computer searches(3) . This region of the MutT
protein has since been shown by NMR and site-directed mutagenesis to be
part of the active site and nucleotide binding site of the MutT
protein(2, 4) . The structure of the MutT protein, as
revealed by heteronuclear multidimensional NMR, shows that this region
forms a novel loop-helix-loop nucleotide binding site not previously
seen in other proteins(5, 6) . In addition, four other
proteins containing homology to the MutT active site have been found to
possess various enzymatic and biological activities. These proteins
include a MutT homologue from Proteus vulgaris(7) , a
human enzyme that degrades the potentially deleterious nucleotide
8-oxo-7-hydro-deoxyguanosine triphosphate
(8-oxo-dGTP)()(8, 9, 10) , the
product of the E. coli orf17 gene, a nucleoside triphosphate
pyrophosphohydrolase(11, 12) , and an E. coli NADH pyrophosphatase(13) .
Here we report the discovery
and characterization of another such enzyme, a GDP-mannose mannosyl
hydrolase. The new enzyme is an E. coli protein coded for by
an open reading frame near the GDP-mannose pyrophosphorylase (cpsB) and phosphomannomutase (cpsG) genes in the
45-min region of the E. coli chromosome. The open reading
frame begins 1.9 kilobase pairs from the start of the putative
GDP-mannose dehydrogenase gene and is therefore referred to as orf1.9 in GenBank accession number
L11721(14) . The protein product of the orf1.9 open
reading frame is listed as yefc in the SwissProt data base.
Protein extracts were analyzed using native and denaturing polyacrylamide gel electrophoresis as described by Laemmli(20) , and proteins in polyacrylamide gels were stained using Coomassie Brilliant Blue R.
Paper electrophoresis of nucleotides was performed in 25 mM citrate buffer, pH 4.9, as described by Markham and Smith(21) . Nucleotides were visualized with ultraviolet light.
High performance anion exchange
chromatography (HPAEC) was done using a Bio-LC (Dionex Corp.,
Sunnyvale, CA) and a CarboPac PA-1 column (4 250 mm) and a
pulsed amperometric detector (PAD-II)(22) . The HPAEC data were
analyzed with A1-450 chromatography software (Dionex).
Protein concentrations were determined using a reagent from Bio-Rad (Hercules, CA) based on the method of Bradford(23) . Bovine serum albumin was used as a standard.
Both radioactive (assay 1) and colorimetric
(assay 2) assays were used to measure the activity of the Orf1.9 E.
coli GDP-mannose hydrolase, each based on the following reaction:
GDP-mannose GDP + mannose.
Figure 1:
Expression
and purification of the Orf1.9 protein. A 15% SDS-polyacrylamide gel
stained with Coomassie Blue. Lane1 contains 4 µg
each of markers ovalbumin (45 kDa), carbonic anhydrase (29 kDa), MutT
(15 kDa), and -lactalbumin (14 kDa). Lanes2 and 3 show the expression of the Orf1.9 protein. HMS174(DE3) cells
containing the plasmid pETorf1.9 were grown in LB-ampicillin (100
µg/ml) medium. Cells (1.5 ml) were harvested before (lane2) and 2 h after (lane3) IPTG
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, and lane3 contains 20 µl of the induced cell extract. Lanes4-7 show stages in the purification of the Orf1.9
protein and contain 6 µg of fractions I-IV,
respectively.
Figure 2:
The induction of the GDP-mannose hydrolase
in crude extracts of HMS174(DE3) cells containing the plasmid
pETorf1.9. Cells were grown to an A of 0.6, and
IPTG was added to a final concentration of 1 mM. Cells were
collected by centrifugation, resuspended in 4 volumes of buffer A, and
sonicated using a Branson Sonifier cell disrupter. After centrifugation
to remove cell debris, enzyme activity in the supernatants was measured
using assay 1 (see ``Methods'').
Figure 3:
The stoichiometry of the Orf1.9 catalyzed
reaction. Purified Orf1.9 protein (0.5 milliunit) was incubated with 2
mM GDP-[H]mannose, 20 mM MgCl
, 80 mM glycine, pH 9.3. The reactions
were terminated by boiling, and aliquots were used to determine free
[
H] mannose (
) using assay 1 (see
``Methods''), inorganic orthophosphate (
) using the
method of Ames and Dubin(25) , and inorganic orthophosphate
after digestion of the products with alkaline phosphatase (
)
using assay 2 (see ``Methods'').
Surprisingly, however, the products of the
reaction were not GMP and mannose-1-phosphate. One of the products was
identified as GDP by paper electrophoresis (Fig. 4). The other
product, mannose, was identified and quantified using HPAEC (Fig. 5). Fig. 5A identifies the Norit
nonadsorbable product of the reaction as mannose based on its
chromatographic characteristics, which are identical to those of
authentic mannose(22) . Fig. 5B shows further
that the accumulation of free mannose is dependent on time of
incubation. The areas under each peak were used to determine the moles
of mannose released. The moles of GDP at each of the time points in Fig. 4B were also determined using assay 2 to measure
alkaline phosphatase labile phosphate. At each time, 2 mol of
phosphate, and hence 1 mol of GDP, were detected for each mole of
mannose produced (data not shown). This agrees with the data presented
in Fig. 3. Thus the equation for the reaction may be written as
follows: GDP-mannose GDP + mannose.
Figure 4:
Identification of GDP product. Reactions
were incubated for 15 min at 37 °C in 50 mM Tris-Cl, pH
9.0, 10 mM MgCl, 4 mM GDP-mannose and
terminated by boiling, and 20 µl were spotted for electrophoresis.
Paper electrophoresis was done according to Markham and
Smith(21) , in 25 mM citrate buffer, pH 4.9, at 1400
volts for 2 h. Lanes1 and 6 contained 100
nmol each of markers GTP, GDP, and GMP. The reaction in lane2 contained no Orf1.9 protein; reactions in lanes3 and 4 contained 2 units and 4 units of enzyme,
respectively; and the reaction in lane5 contained
the same components as lane4 without
MgCl
. Nucleotides were visualized with UV
light.
Figure 5:
Identification of mannose product. A, reactions were done at 37 °C in 40 mM glycine,
pH 9.3, 10 mM MgCl, and were terminated by the
addition of 1 volume of 18% Norit in 0.1 N HCl before (
)
and 20 min after (
) addition of purified Orf1.9 protein (1.1
milliunits). After centrifugation, 50 µl of the supernatant was
evaporated to dryness and dissolved in 500 µl of H
O. 50
µl were analyzed by HPAEC on a CarboPac PA1 column (4
250
mm) eluting with 16 mM NaOH(22) . The Norit
nonadsorbable product eluted at the the same time as a mannose standard
(
) (1 nmol). B, reactions were done as described above
and were terminated by boiling after 10 min (--), 20
min(- - -) or 30 min (
),
evaporated to dryness, and dissolved in 2.5 ml of H
O. 50
µl were chromatographed as described above. (Mannose-1-phosphate is
not eluted from the column under these
conditions.)
A
kinetic analysis of substrate titrations with GDP-mannose and
GDP-glucose is presented in Table 2. Although the enzyme has a
4.7-fold higher V for GDP-glucose than for
GDP-mannose, the K
for GDP-mannose is 6.3-fold
lower, resulting in a somewhat higher overall catalytic efficiency (V
/K
) for GDP-mannose.
Like other enzymes in this class, the E. coli GDP-mannose
mannosyl hydrolase absolutely requires divalent metal cations for
activity as shown in Fig. 4, lane5, where
MgCl is absent from the reaction mixture. The only metals
tested that effectively activated the enzyme were Mg
and Mn
. Ca
,
Zn
, and Co
supported 4, 3, and
<1%, respectively, of the activity of Mg
.
Another property that this enzyme shares in common with several enzymes containing the consensus region is its pH versus rate profile. The enzyme has an alkaline pH optimum at pH 9.3, which is similar to the pH optima of the MutT enzyme(26) , the Orf17 enzyme (12) , and the NADH pyrophosphatase(6) .
Figure 6: Alignment of the Orf1.9 amino acid sequence with other enzymes containing homology to the MutT active site. The amino acid sequences of the MutT and Orf1.9 proteins were aligned using the computer program CLUSTAL W 1.5(27) . Identical amino acids are boxed in black, and similar amino acids are noted with grayboxes. The sequences aligned with the Orf1.9 GDP-mannose hydrolase are as follows: MutT, the MutT protein from E. coli(42) ; Orf1.7, nucleoside triphosphate pyrophosphohydrolase from E. coli(11) , NADHase, the NADH pyrophosphatase from E. coli(13) ; MutT(PV), the MutT homologue from P. vulgaris(7) ; MutX, the MutT homologue from S. pneumoniae(2) ; and 8-oxo-dGTPase, an enzyme from human cells that degrades 8-oxo-dGTP(9) . Gaps in the protein sequences required to optimize the alignment are represented by hyphens.
The majority of the enzymes that hydrolyze nucleotide sugars
are pyrophosphatases(28, 29) ,
phosphorylases(30, 31) , or
pyrophosphorylases(32) . Unlike the enzyme described here,
these enzymes release sugar-1-phosphates rather than free sugar. Only
one other enzyme has been reported that releases a free sugar from the
nucleotide sugar, a GDP-glucose glucohydrolase (EC 3.2.1.42) isolated
from yeast(33) . This enzyme differs from E. coli Orf1.9 in that it has no activity on GDP-mannose and does not
require divalent metal ions for activity(33) . We suggest the
name GDP-mannose mannosyl hydrolase for the E. coli enzyme to
point out this difference. This name also conforms to the standard
practice of naming the enzyme after the substrate with the lowest K. The possible biological roles of nucleotide
sugar hydrolases were discussed by Sonnino et
al.(34) , and similar roles are conceivable for the E.
coli enzyme. The enzyme could participate in the regulation of
cell wall biosynthesis by influencing the concentration of GDP-mannose
or GDP-glucose in the cell. Because the enzyme shows little activity on
GDP-fucose and is present in the cps gene cluster that may be
involved in the synthesis of GDP-fucose(14) , another role of
the Orf1.9 protein could be to degrade GDP-mannose and GDP-glucose,
diverting the GDP to the synthesis of GDP-fucose as required.
Our
interest in the enzyme, however, is mainly focused on its similarity to
the MutT protein which, when defective, increases the simultaneous rate
on the order of 1000-fold(35) . Since we noticed that the MutT
catalytic core was widely distributed throughout nature(2) , we
have begun to characterize other proteins that share this common motif
in order to uncover its biochemical mechanism and to catalogue the
physiological function of proteins harboring the signature sequence.
These MutT-like proteins may be of special interest, because there have
been reports of suppressors of the mutT phenotype in E. coli(36, 37) . Such
genes could code for proteins with similar functions to MutT that could
also be involved in the maintenance of the fidelity of DNA replication.
To date, the enzymatic activities of seven proteins containing the
GX(I/L/V)(E/Q)(X)ET(X)
R(X)
E(X)
(I/L)
signature have been identified, namely those of MutT from E.
coli(38) , the homologous MutT from P.
vulgaris(7) , the MutX protein from S.
pneumoniae(2) , the human 8-oxo-dGTPase(9) , the
Orf17 protein(11) , the E. coli NADH
pyrophosphatase(13) , and the E. coli GDP-mannose
hydrolase. Table 3summarizes the reactions catalyzed by these
proteins. The first five of these enzymes (listed in the order of their
discovery) are nucleoside triphosphatases. The four that have been
characterized have been shown to hydrolyze the linkage between the
and
phosphates forming a nucleoside monophosphate and
inorganic pyrophosphate. Based on our recent discovery of the NADH
pyrophosphatase (13) and the GDP-mannose hydrolase reported
herein, we can narrow down the features of the substrate required for
recognition by the catalytic region. All active substrates are
derivatives of nucleoside pyrophosphates, and therefore we hypothesize
that this signature designates a unique nucleoside diphosphate binding
and catalytic domain.
In addition to the enzymes mentioned here, recent computer searches of nucleic acid and protein data bases have revealed that several viral, prokaryotic, and eukaryotic proteins also share homology to this motif. These include proteins from African swine fever virus, vaccinia virus, fowlpox virus, variola virus, Streptomyces ambofaciens, proteins coded for by the antisense RNA of Xenopus laevis and human basic fibroblast growth factors(2, 3) , and a protein from Chilo iridescent virus(39) . The results presented here should be considered when speculating about the cellular functions of these proteins. The observations that both an NADH pyrophosphatase and a GDP-mannose hydrolase contain this consensus sequence indicate that the catalytic region designated by this motif is not confined to enzymes having nucleoside triphosphatase activity. Instead, it has been conserved during evolution and adapted to participate in diverse metabolic reactions involving the cleavage of substrates containing a pyrophosphate group linked to a nucleoside.
Structural analysis of
the MutT protein has already revealed that the conserved region folds
to form a unique nucleotide binding motif(5, 6) and
that amino acids in the signature sequence are in intimate contact with
bound nucleotides(4) . Further studies are designed to uncover
the roles of specific amino acids in this domain. Detailed structural
and enzymatic analyses of these other MutT-like proteins would likewise
be of interest to determine whether or not the consensus sequence
GX(I/L/V)(E/Q)(X)ET(X)
R(X)
E(X)
(I/L)
forms a similar nucleotide binding site in these proteins as well. Thus
far, all seven of the characterized proteins containing the above
signature sequence hydrolyze nucleoside pyrophosphate compounds,
suggesting that the other uncharacterized proteins sharing this
signature sequence have similar activities. These include proteins of
unknown function from a wide variety of organisms, ranging from viruses
to humans. It is tempting to speculate that the identification and biochemical characterization of consensus sequences such as
these in the rapidly expanding data banks will, in the future,
facilitate the determination of protein function from sequence data
alone.