Glucose phosphorylation is catalyzed by a family of structurally
related hexose phosphotransferases. These enzymes can be divided into
two general groups: the Class I enzymes, which include hexokinase I,
II, and III, are larger (
100 kDa) and have a high affinity for
glucose (K
= 20-130
µM)(1, 2) ; the Class II enzyme, which is
called hexokinase IV or glucokinase, is smaller (
50 kDa) and
displays a low affinity for glucose (K
= 5-8 mM) (reviewed in (3) ).
Comparison of the deduced amino acid sequences of mammalian liver
glucokinases with that of hexokinase I, II, and III (4) revealed that the hexokinases are essentially dimers of
glucokinase, thus providing evidence for the hypothesis that the
hexokinases have evolved from a primordial glucokinase-like gene by
gene duplication(1, 4, 5) . However, from a
phylogenetic hexokinase family tree constructed by comparing 60
sequences of sugar kinases, Bork et al.(6) observed
that glucokinases appear in three clusters in separate branches, where
(i) the mammalian glucokinases formed one cluster, (ii) the yeast
glucokinase appeared to be clustered with yeast hexokinases rather than
with mammalian glucokinases, and (iii) bacterial glucokinases from Zymomonas mobilis and Streptomyces coelicolor were
grouped with the Zymomonas fructokinase. These authors
concluded that a divergent evolutionary relationship between these
glucokinases was unlikely. Rather, they argued that evolutionary
convergence to glucose specificity must have occurred independently in
mammals, yeast, and bacteria.
Our interest in the evolutionary
origin of the glucokinases led us to investigate the properties of
polyphosphate glucokinases (Poly(P)-glucokinase) (
)from
different sources. Inorganic polyphosphates (poly(P)s) are linear
polymers of orthophosphate linked by phosphoanhydride bonds. These
polymers have been found in almost all species, and their proposed
biological functions have been reviewed in
detail(7, 8, 9, 10) . One of the
poly(P)-utilizing enzymes, Poly(P)-glucokinase (EC 2.7.1.63), on the
other hand, has been found only in certain
bacteria(7, 11) . This Poly(P)-glucokinase, which was
first observed in the Mycobacterium phlei by
Szymona(12) , is an unusual glycolytic enzyme that utilizes
poly(P) or ATP as the phosphoryl donor to phosphorylate glucose. We
previously reported the purification of this enzyme from Propionibacterium shermanii(13) and Mycobacterium
tuberculosis H
Ra (14) and demonstrated that
the poly(P)- and ATP-dependent glucokinase activities were catalyzed by
a single enzyme. Poly(P)-glucokinase from M. tuberculosis also
utilized GTP, UTP, and CTP as the phosphoryl donors(14) . In
addition, kinetic studies with the P. shermanii enzyme
suggested that ATP and poly(P) have different binding
sites(13) . It has been hypothesized (13) that glucose
phosphorylation in the microorganism may have originally been mediated
by poly(P), and when ATP became available in the environment, a
transition was made to utilize the latter phosphoryl donor by the
glucokinases. Thus, this bifunctional Poly(P)-glucokinase could
represent an ``intermediate'' in the evolution of
glucokinases, especially in prokaryotic cells.
In attempts to
identify the structural and functional domains of the
poly(P)-glucokinase, we have cloned, sequenced, and expressed the
Poly(P)-glucokinase gene (ppgk) from M. tuberculosis H
Rv. The expressed and purified recombinant
Poly(P)-glucokinase (re-Poly(P)-glucokinase) from Escherichia
coli, showed that the cloned ppgk gene encodes a single
polypeptide chain containing both the poly(P)- and ATP-dependent
glucokinase activities. Through sequence alignment with other
glucokinases, phosphate binding motifs were found to be conserved in
the Poly(P)-glucokinase and other prokaryotic glucokinases. In
addition, a putative poly(P) binding site for the Poly(P)-glucokinase
is proposed.
EXPERIMENTAL PROCEDURES
Materials
E. coli strains Y1090 and
DH5
were purchased from Life Technologies, Inc. E. coli strains BL21 and BL21 pLysS were purchased from Novagen, Inc. M. tuberculosis H
Ra was a gift from Dr. O.
Szymona (Institute of Basic Chemical Sciences, Medical School of Lubin,
Lubin, Poland). The
gt-11 genomic library of M. tuberculosis ``Erdman,'' TMC 107, was a gift from Dr. J. Ellner (15) (Case Western Reserve University). Immobilon-NC transfer
membranes (HATF) were from Millipore Corp.
[
-
P]CTP (3000 Ci/mmol) was from Amersham
Corp.
[
S]deoxyadenosine-5`(
-thio)triphosphate was
from DuPont NEN. Random primed DNA labeling kit, acrylamide, N-methylenebisacrylamide, isopropylthiogalactoside,
endoproteinase Arg-C, trypsin, urea, and all restriction enzymes were
from Boehringer Mannheim. Oligonucleotides were custom made by Midland. Staphylococcus aureus V
protease was from Miles
Scientific. ATP, glucose, glucose-6-phosphate dehydrogenase, and sodium
phosphate glass (Type 35) were from Sigma. All other chemicals were of
reagent grade.
Amino Acid Sequences of Poly(P)-glucokinase Peptides and
Design of Degenerate Primers
Approximately 100 µg of
Poly(P)-glucokinase was digested with endoproteinase Arg-C, and the
peptides were fractionated by Tricine-SDS-PAGE (16) followed by
electroblotting onto a polyvinylidene difluoride membrane(17) .
The membrane was then stained with Coomassie Brilliant Blue R-250 and
destained. A peptide with a migration corresponding to 27 kDa (see Table 1, peptide number 1) was excised and subjected to
N-terminal sequencing on an Applied Biosystem Model 470 sequencer at
the core facility of Case Western Reserve University. Because the
native enzyme migrated as a 33-kDa protein(14) , this peptide
sequence was expected to be close to the N terminus of the enzyme.
Therefore, we designed degenerate oligonucleotides,
5`-GGAATTCCTT(T/C)GGIGTIGA(T/C)GTNGGNGG-3`, as the sense primers based
on the amino acid sequence FGVDVGG. Internal peptides were obtained by
digesting the enzyme with V
-protease or trypsin and
separating the digest on a Synchropak C
column (RP-8, 25
0.46 cm) using reverse-phase high performance liquid
chromatography. Peptides were eluted with a linear gradient using 0.1%
aqueous trifluoroacetic acid (solvent A) and 0.1% trifluoroacetic acid
in CH
CN (solvent B). Peptide elution was monitored at 220
nm, collected manually, and subjected to N-terminal sequencing. The
antisense primers sequences, based on peptide number 4 sequence
EEHYGAG, are 3`-CT(T/C)CT(T/C)GA(A/G)AT(G/A)CCICGNCCICCTTAAGG-5`. For
both primers, letter N represents the position where ATGC were at the
same place. Nucleotide, inosine (I), was sometimes used to substitute
ATGC, reducing the degeneracy to 64-fold for both primers.
Genomic Cloning of Poly(P)-glucokinase
Gene
Degenerate sense primers (28-mers) and the degenerate
antisense primers (29-mers) corresponding to the sequences of two
peptides (see Table 1, numbers 1 and 4) of Poly(P)-glucokinase
from M. tuberculosis H
Ra were used as primers in
PCR. To isolate the DNA, approximately 10 g of M. tuberculosis H
Ra cells were freeze-thawed twice and resuspended in
35 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH
7.4), and 2.5 ml of lysozyme (10 mg/ml) was added, mixed, and incubated
for 1 h at 37 °C. To this, 3.5 ml of 10% SDS and 300 µl of
proteinase K (10 mg/ml) were then added, and the solution was incubated
for 30 min at 65 °C. Purification of genomic DNA was then carried
out by published methods(15) .Around 100 ng of purified
genomic DNA was used as a template in a PCR reaction. Sixteen different
buffer conditions, with variable Mg
concentrations
and pH values, were set up based on the PCR Optimizer kit (Invitrogen),
and 30 cycles of 95 °C (2 min), 55 °C (3 min), and 72 °C (3
min) were performed. A major 365-bp product was observed on a 1%
agarose gel under the following conditions: Mg
(2.0
mM), Tris-HCl (60 mM, pH 9.0), and
(NH
)
SO
(15 mM). This
fragment was gel-purified and subcloned into the vector, pBluescript
SK
(Stratagene, La Jolla, CA). The resulting plasmid
was designated as pBS-PCR-1. Sequence analysis of pBS-PCR-1 indicated
that the deduced sequence encoded the amino acid sequences of the two
isolated peptides (numbers 1 and 4) as shown in Table 1. A high
specific activity probe was generated using 25 ng of this 365-bp
fragment as a template and the Random Primed DNA labeling kit and 5
µCi of [
-
P]CTP. The resulting
P-labeled probe was used to screen the
gt-11 genomic
library of M. tuberculosis H
Rv. Around 6
10
plaques were screened on four duplicate filters.
Hybridizations were performed at 65 °C overnight in 6
SSC
buffer (20
SSC = 3 M of NaCl and 0.3 M sodium citrate), 1% (w/v) bovine serum albumin, and 0.02% (w/v)
SDS. The filters were then washed three times with 2
SSC
containing 0.1% SDS at room temperature for 5 min each and with 1
SSC containing 0.1% SDS at 65 °C twice for 2 h each.
Autoradiographs were prepared by exposure to x-ray film (Kodak X-OMAT
AR) at room temperature for 4 h or at -70 °C overnight.
Eighteen positive clones were detected in the primary screening.
After tertiary screening, only seven plaques remained positive. These
phages were enriched separately on 150-mm LB medium plates, and the
phage-DNAs were purified, digested with EcoRI, and separated
on a 0.8% agarose gel. From these seven
-phage DNAs, eight
fragments below 10 kb were observed that represented the inserts of M. tuberculosis H
Rv DNA. The fragments were then
gel-purified and subcloned into pBluescript. The resulting plasmids
were designated as pBS-PPGK 1 to 8.
Reselection of pBS-PPGK Clones
Two primers based
on the 365-bp insert in pBS-PCR-1 were synthesized to determine which
of the 8 pBS-PPGK plasmids was best suited for further analysis. Primer
I contains 5`-end sequences (5`-CAGCGGGATCAAGGGCG-3`) and primer II
contains 3`-end sequences (5`-TGCGGCGTCTGCGTCGTT-3`). Thermal cycles of
PCR reactions were one cycle of 94 °C (2 min), 30 cycles of 94
°C (1 min), 60 °C (2 min), and 72 °C (1 min), and one cycle
of 72 °C (7 min). This reaction was designed to produce a 280-bp
fragment. Two out of the eight clones yielded the expected result, and
one, pBS-PPGK-7, was selected for further analysis.
Southern Blotting Analysis
pBS-PPGK-7 plasmid was
digested with different restriction enzymes and separated on a 1%
agarose gel in a Tris-boric acid-EDTA system. The DNA fragments were
transferred onto a nylon membrane by the capillary method(18) .
Generation of the radioactive probe, hybridization conditions, and
autoradiographs were the same as described above.
Sequence Analysis
Nucleotide sequence analysis was
performed by using the Sanger dideoxy chain termination method
(Sequenase Version 2.0, U. S. Biochemical Corp). DNA template and
sequencing primer were incubated at 90 °C for 2 min and transferred
to 37 °C for 15 min. The resulting sequences were used to design
additional primers in a primer-walking strategy. For pBS-PPGK-7 gene,
1100-bp was sequenced from both strands (see Fig. 1) including
795-bp representing the ppgk open reading frame as shown in Fig. 2.
Figure 1:
Partial restriction map of the
pBS-PPGK-7 clone and the DNA sequencing strategy. The restriction map
of pBS-PPGK-7 clone carrying the 2.3-kb DNA fragment from M.
tuberculosis H
Rv containing the region that
hybridized with random primed probes from a 365-bp PCR product. Its
orientation with respect to T7 promoter of pBluescript is shown. An
expanded map containing Poly(P)-glucokinase open reading frame with
sequencing strategy is shown below the restriction map. The nucleotide
sequence of 1.1-kb fragment that was obtained is represented by the
length and the direction of the arrows. The restriction
enzymes are, X, XhoI; S, SalI; P, PstI; N, NcoI; E, EcoRI; N
, NotI; EV, EcoRV; P
, PvuII.
Figure 2:
Nucleotide sequence and derived amino acid
sequence of Poly(P)-glucokinase gene from M. tuberculosis H
Rv. The nucleotide sequence was obtained from
pBS-PPGK-7 (shown in Fig. 1). A possible initiator methionine is
labeled +1. The deduced amino acid sequence is shown below the nucleotide sequence. A putative Shine-Dalgarno
sequence for translation initiation is underlined. Peptide
sequences (shown in Table 1) obtained from M. tuberculosis H
Ra are also aligned with those from
H
Rv.
Assay of Poly(P)-glucokinase Activity
The
enzymatic activity was measured by coupling the formation of
glucose-6-phosphate to glucose-6-phosphate dehydrogenase and monitoring
the formation of NADH spectrophotometrically at 340 nm(14) .
One unit of glucokinase activity is defined as that amount of enzyme
that catalyzes the formation of 1 µmol of glucose-6-phosphate/min
at 30 °C.
Expression of Recombinant Poly(P)-glucokinase in E. coli
BL21 and BL21 pLysS Strains
The construction of the expression
vector, pET-23a-PPGK, is shown in Fig. 3. This plasmid was
transformed into BL21 and BL21 pLysS E. coli strains
separately, and control experiments were performed by transforming with
pET-23a without the ppgk gene. The BL21 cells were grown in LB
medium containing carbenicillin (50 µg/ml) at 37 °C, and the
BL21 pLysS cells were grown in LB medium containing carbenicillin (50
µg/ml) and chloramphenicol (34 µg/ml) at 37 °C. When E.
coli reached an A
of approximately
0.8-1, IPTG was added to a final concentration of 0.4
mM, and 2 ml of culture were removed after 1, 2, 4, and 6 h
after induction. The cells were harvested, centrifuged, and resuspended
in 150 µl of buffer containing 50 mM glucose, 1 mM MgCl
, 0.5 mM EDTA, 0.5 mM
-mercaptoethanol, and 10 mM Tris-HCl (pH 7.4). The
cells were sonicated followed by centrifugation at 12,000
g for 15 min. The supernatant was removed, and the pellet was
resuspended in the buffer described above. Expression of the
re-Poly(P)-glucokinase was examined by monitoring the poly(P)-dependent
glucokinase activity.
Figure 3:
Construction of sequence and expression
plasmid. The ppgk gene contained in a 2.3-kb EcoRI
fragment from pBS-PPGK 7 was PCR-amplified with two synthetic primers,
Primer I, 5`-GGAATTCCATATGACCAGCACCGGCCCCGA-3`, and Primer II,
5`-GGAATTCCATATGGTAACTTTACTGCGCACGAG-3`. The primers were designed to
carry EcoRI and NdeI sites on both 5` end. Thermal
cycles of PCR were one cycle of 94 °C (2 min), 30 cycles of 94
°C (1 min), 60 °C (2 min), and 72 °C (1 min), and one cycle
of 72 °C (7 min). Vent
DNA polymerase (from New England
Biolabs) was used. This amplified product was subcloned into EcoRI site of pBluescript plasmid to confirm the sequence
after PCR. This ppgk gene was then subcloned into NdeI site of the expression vector pET-23a (Novagen). Correct
orientation of insertion was determined by SalI and PvuII digestion. The resulting plasmid was designated
pET-23a-PPGK.
Purification of Recombinant Poly(P)-glucokinase from E.
coli
All purification steps were carried out at 4 °C unless
otherwise stated, and all buffers contained 50 mM glucose, 1
mM MgCl
, 0.5 mM EDTA and 0.5 mM
-mercaptoethanol. BL21 pLysS cells that carried the
pET-23a-PPGK plasmid were grown in LB medium containing 50 µg/ml
carbenicillin and 34 µg/ml chloramphenicol at 37 °C. When the
optical density (A
) reached 0.6-0.8,
IPTG (final concentration, 0.4 mM) was added, and cells were
permitted to grow for another 3 h. The cells (
18 g) were pelleted
by centrifugation, resuspended in 25 ml of 10 mM Tris-HCl (pH
7.4), and sonicated 3 times for 5 min each. Streptomycin sulfate (final
concentration, 6%, w/v) was added, mixed for 30 min, and centrifuged at
20,000
g in a SS34 rotor. Supernatant was collected,
mixed with ammonium sulfate (final concentration, 30%, w/v) for 30 min,
and centrifuged as above. The supernatant was brought to 70% (w/v)
saturation with ammonium sulfate for 30 min and centrifuged as above.
The pellet was collected, resuspended in 18 ml of 50 mM potassium phosphate (KPi) buffer, and dialyzed against 4 liters of
50 mM KPi (pH 6.8) buffer overnight. The dialysate was pooled
and applied to a 60-ml phospho-cellulose (P-11) column (Whatman), and
bound proteins were eluted with a linear gradient of 50 to 500 mM KPi buffer (pH 6.8). Poly(P)-glucokinase activity was detected
between 0.3 and 0.35 M KPi gradient. The
re-Poly(P)-glucokinase containing fractions were dialyzed overnight
against 4 liters of 50 mM KPi buffer and applied to a
hydroxyapatite column. The bound protein was then eluted with a linear
gradient of 50 mM to 500 mM KPi buffer. The
re-Poly(P)-glucokinase eluted between 0.3 and 0.35 M KPi
gradient. The re-Poly(P)-glucokinase containing fractions were then
dialyzed against 10 mM Tris-Maleate (pH 6.8) buffer overnight
and applied to an Affi-Gel Blue column (Bio-Rad). The bound protein was
then eluted with the same buffer containing 5 mM (in terms of
orthophosphate concentration) polyphosphate (Type 35) and 2 mM MgCl
. The re-Poly(P)-glucokinase containing fractions
were pooled, concentrated using Amicon ultrafiltration apparatus
equipped with a YM-10 membrane, and then stored at -20 °C.
RESULTS
Cloning of the Poly(P)-glucokinase Gene
Our
cloning strategy for the ppgk gene involved the generation of
the probe employing PCR with the genomic DNA template, followed by
screening a M. tuberculosis genomic library and identifying a
full-length clone. Initially two degenerate primers were designed based
on amino acid sequences of two peptides (Table 1, numbers 1 and
4). These two primers together with genomic DNA were used in a PCR to
generate a 365-bp product, which was used as a probe to screen the
genomic library of M. tuberculosis. Out of the 60,000 plaques,
seven clones were identified by plaque hybridization. Reselection of
pBS-PPGK plasmids 1-8 by an additional round of PCR, as described
under ``Experimental Procedures,'' yielded two plasmids with
the expected 280-bp product, and they were designated as pBS-PPGK-7 and
pBS-PPGK-8. In order to position the open reading frame of the ppgk gene in the pBS-PPGK-7 insert, this plasmid was digested with
different restriction enzymes, and the digested fragments were
separated on a 1% agarose gel followed by Southern blot analysis with
the 365-bp PCR product as a probe. As shown in Fig. 1, the
2.3-kb EcoRI insert was found to contain the open reading
frame of the ppgk gene. An expanded scheme of the sequencing
strategy is also shown below the restriction map in Fig. 1. The M. tuberculosis H
Rv ppgk gene sequence
and the deduced amino acid sequence are shown in Fig. 2. The
peptide sequences that were obtained from M. tuberculosis H
Ra (Table 1) are also aligned below that
deduced from the H
Rv ppgk gene sequence. The open
reading frame of the gene consisted of 795 bases that predicted a
polypeptide of 265 amino acids with a calculated molecular mass of
27,400 Da. In common with other M. tuberculosis genes(19, 20, 21) , the ppgk gene showed a high G+C content (64.5%), and analysis of codon
usage bias showed a strong preference of G and C in the third base
position. A putative Shine-Dalgarno sequence (GAGGAG) was also
identified in the nucleotide sequence upstream from the proposed
initiation codon (labeled as +1). However, a typical E.
coli-like promoter consensus sequence was not apparent at the
-10 or -35 nucleotide positions.
Expression of Recombinant Poly(P)-glucokinase in E.
coli
The construction of the expression plasmid, pET-23a-PPGK,
is diagrammed in Fig. 3. Two E. coli strains, BL21 and
BL21 pLysS, carrying this plasmid, were tested for the expression of
re-Poly(P)-glucokinase. As shown in Table 2, when BL21 pLysS or
BL21 were used as hosts and transformed with pET-23a vector by itself,
Poly(P)-glucokinase activity was not detected in cell lysates as
determined by the enzymatic activity assay, using poly(P) as a
substrate, as described under ``Experimental Procedures.''
When the BL21 pLysS was transformed with pET-23a-PPGK plasmid,
Poly(P)-glucokinase activity was detected only in those cells that were
induced with IPTG. The re-Poly(P)-glucokinase was expressed as a
soluble cytosolic protein, and no Poly(P)-glucokinase activity was
found in the cell pellets. The ATP-dependent glucokinase activity was
also determined in these cells. As shown in Table 2, the specific
activity of ATP-dependent glucokinase was about two times higher if the
cells were induced by IPTG. The increased specific activity is likely
due to the expression of the re-Poly(P)-glucokinase encoded in the
pET-23a-PPGK plasmid. Similar results were obtained when a BL21 strain
was used, although some amount of re-Poly(P)-glucokinase was expressed
without IPTG induction. This leakage is common in BL21 cells as
described by the manufacturer (Novagen). Hence, the BL21 pLysS strain
was used for the expression of this recombinant enzyme.
Purification of Recombinant Poly(P)-glucokinase from E.
coli
The purification procedures are described under
``Experimental Procedures,'' and the results are shown in Table 3. As calculated from the purification table, the
re-Poly(P)-glucokinase was not expressed at high levels (0.4% of total
cellular proteins). The re-Poly(P)-glucokinase was purified 189-fold to
near homogeneity, as judged by SDS-PAGE (Fig. 4), with a 18%
recovery. The ATP-dependent glucokinase activity was co-purified with
poly(P)-dependent glucokinase activity during the course of
purification with a constant ratio of poly(P)-dependent activity to
ATP-dependent activity (around 3.5 except in the crude extract stage).
When the purified enzyme was chromatographed into a gel filtration
column (TSK-G3000SW) or a C
reverse-phase column, only a
single peak was observed (data not shown). In addition, the single peak
from the gel filtration column was found to contain both poly(P)- and
ATP-dependent glucokinase activities. Hence, the minor band seen in lane 7 of Fig. 4might be an artifact from the SDS
preparation as was observed in other cases(22) . Similar to
Poly(P)-glucokinase from M. tuberculosis H
Ra, the
purified re-Poly(P)-glucokinase possessed both the poly(P)- and
ATP-dependent glucokinase activities. The specific activities of this
purified enzyme were found to be 203 units/mg and 61 units/mg for the
poly(P)- and ATP-dependent glucokinase activities, respectively (Table 3). These values are similar to those obtained for the
Poly(P)-glucokinase from the H
Ra strain (14) .
Thus, these results support our previous finding that both activities
are catalyzed by a single protein based on a number of different
criteria(14) . Although the calculated molecular mass is 27,400
Da, the re-Poly(P)-glucokinase migrated as a 33-kDa protein on SDS-PAGE (Fig. 4, lane 7), which is identical to the migration
of the native enzyme from the H
Ra strain (Fig. 4, lane 1). The difference in the observed and calculated
molecular mass may be due to the presence of a cluster of charged
groups (amino acids 188-200 and 222-229, Fig. 2),
which could cause anomalous migration of the enzyme by SDS-PAGE. Such
an effect of clusters of charged groups on the mobility of proteins on
SDS-PAGE has been observed with the RNA-binding
protein(23, 24) .
Figure 4:
SDS-polyacrylamide gel electrophoresis of
re-Poly(P)-glucokinase from E. coli. The 12.5% SDS-PAGE was
carried out by the method of Laemmli(37) . Protein was
visualized by staining with Coomassie Brilliant Blue R-250 and
destaining with methanol/acetic acid. Lane 1,
Poly(P)-glucokinase purified from M. tuberculosis H
Ra(14) ; lane 2, molecular mass
standards from Amersham Corp. were 200, 94, 67, 43, 30, 23, and 14 kDa
(from top to bottom); Lanes 3-7,
re-Poly(P)-glucokinase in crude extract (lane 3), 30-70%
ammonium sulfate precipitate (lane 4), eluant from P-11 column (lane 5), eluant from hydroxyapatite column (lane 6),
or eluant from Affi-Blue gel column (lane
7).
DISCUSSION
Previously, we reported the purification of
Poly(P)-glucokinase from M. tuberculosis H
Ra to
homogeneity and characterized some of its biochemical
properties(14, 25) . In this study, we report the
cloning and sequencing of the ppgk gene. The cloned ppgk gene contains a full length of open reading frame, and several
peptide sequences of the purified Poly(P)-glucokinase from the
H
Ra strain were identified in the deduced amino acid
sequence. The expressed re-Poly(P)-glucokinase purified from E.
coli contained both the poly(P)- and ATP-dependent glucokinase
activities. On the basis of these results, we conclude that this clone
represents the ppgk gene of M. tuberculosis H
Rv, and its translated polypeptide is
Poly(P)-glucokinase. The cloning and expression of the mycobacterial
Poly(P)-glucokinase in E. coli has enabled us to obtain
purified enzyme in a relatively short time, considering the slow
growing nature of M. tuberculosis. The identification of two
conserved phosphate binding domains through sequence alignment (as see
below) with other glucokinases has paved the way for future studies to
test the putative functional residues through site-directed
mutagenesis.
We demonstrated previously that the Poly(P)- and
ATP-glucokinase activities from both M. tuberculosis and P. shermanii were catalyzed by a single
enzyme(13, 14) . Although the protein chemical
evidence was compelling, earlier biochemical data on the
Poly(P)-glucokinase from different sources raised the possibility that
at least in some cases, the poly(P)- and ATP-dependent glucokinase
activities were catalyzed by distinct
enzymes(26, 27, 28, 29, 30) .
However, the observation that the M. tuberculosis ppgk gene
expressed in E. coli displayed both the poly(P)- and
ATP-dependent activities offers conclusive evidence at the DNA level
that the two activities are catalyzed by a single protein.
Characterization of the Poly(P)-glucokinase
Gene
Although a potential Shine-Dalgarno region was identified 5
bp upstream of the proposed translation initiation site, an E.
coli-like consensus promoter region was not obvious. Preliminary in vitro transcription experiments using E. coli RNA
polymerase and the ppgk gene template (nucleotide positions
-78 to 97) did not yield any transcribed mRNA. Other studies on
the promoter regions of a number of M. tuberculosis genes
including 85A antigen(31) , cpn 60(32) , the
Mycobacterium bovis Bacillus Calmetle-Guèrin hsp 60(33) , Bacillus
Calmetle-Guèrin mph 70(34) , and
the Mycobacterium smegmatis ask(35) genes showed that E. coli-like promoter regions were also absent in these genes.
Hence, in order to identify the promoter region of the M.
tuberculosis ppgk gene, we will have to subclone the potential
promoter region from the ppgk gene into a Mycobacterium-E.
coli shuttle vector followed by transformation into the
nonpathogenic mycobacterial strain.The ppgk gene sequence
predicts a polypeptide of 265 amino acids with a potential
translational initiator methionine. One unknown polypeptide sequence
(gi 699175) from a Mycobacterium leprae B1764 cosmid was found
to have 71% sequence identity with that of the Poly(P)-glucokinase.
After omitting the first 68 amino acids from the N-terminal of the open
reading frame of this gi 699175 from M. leprae for
comparisons, these two proteins show 83% sequence identity. Hence, gi
699175 from M. leprae is likely to be Poly(P)-glucokinase,
with an open reading frame encoding a protein of 34 kDa. Because the
function of the gi 699175 has not been characterized, we did not
include it in the sequence homology comparisons shown in Table 4.
Identification of Phosphate Binding Domains of
Poly(P)-glucokinase and Other Prokaryotic Glucokinase
Bork and
co-workers(6, 36) found that the three-dimensional
structures of actin, hexokinase, and Hsp70 protein families contained
common motifs interacting with the ATP molecule, which are the
``Phosphate-1'' and ``Phosphate-2'' motifs
contacting the
- and
-phosphates of ATP and the
``Connect-1'' and ``Connect-2'' motifs at the
interface between the subdomains. Residues in these motifs involved in
the interaction of ATP are highly conserved in many glucokinases; they
are Asp and Gly (in Phosphate-1), Asp (in Connect-1), Gly and Thr (in
Phosphate-2), and Gly (in Connect-2). As shown in Table 4,
analysis of the deduced amino acid sequences of the ppgk gene
shows that this enzyme contains regions that are homologous to
Phosphate-1 and Phosphate-2 regions of yeast glucokinase. Further
sequence alignment analyses on other prokaryotic glucokinases sequences (Table 4) also indicate the presence of phosphate binding motifs.
The homologies within the Phosphate-1 motif and Phosphate-2 motif were
analyzed by the Multiple Alignment Construction and Analysis Workbench
program (BLOSUM-62) and were found to be statistically significant with p values of 4.5
10
and 3.8
10
, respectively. This result implies that these
phosphate binding sites are conserved from eukaryotic hexokinases to
prokaryotic glucokinases.The identity of sequences involved in
polyphosphate binding is less clear. Previously, we demonstrated that
tryptophans in the peptide, KNDWTYPKWAKQ, of Poly(P)-glucokinase from
H
Ra strain were found to be selectively oxidized by N-bromosuccinimide with concomitant loss of enzymatic
activity(25) . Tetrapolyphosphate or long chain polyphosphate
substrate afforded protection against this oxidation and the loss of
activity. Residues 177-216 of the Poly(P)-glucokinase sequence
from H
Rv encodes a closely related sequence, RKDWSYARWSEE.
Hence this region might be a binding site for polyphosphate, in
addition to Phosphate 1 and 2, which specifically enables the
Poly(P)-glucokinase to utilize polyphosphates. Several charged groups
around this region, Lys
, Glu
,
Lys
, Asp
, Lys
, and
Lys
, may fulfill the requirement for poly(P) binding.
However, conclusive evidence for a poly(P) binding site will have to
await site-directed mutagenesis of residues in this region.