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Protein Engineering vol.11 no.12 pp.1219–1227, 1998
Thermostable glycerol kinase from a hyperthermophilic archaeon:
gene cloning and characterization of the recombinant enzyme
Yuichi Koga1, Masaaki Morikawa1, Mitsuru Haruki1,
Haruki Nakamura2, Tadayuki Imanaka3 and
Shigenori Kanaya1,4
1Department
of Material and Life Science, Graduate School of Engineering,
Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, 2Department of
Bioinformatics, Biomolecular Engineering Research Institute, 6-2-3
Furuedai, Suita, Osaka 565-0874 and 3Department of Synthetic Chemistry
and Biological Chemistry, Graduate School of Engineering, Kyoto
University, Kyoto 606-8501, Japan
4To
whom correspondence should be addressed. E-mail:
[email protected]
The Pk-glpK gene, which encodes glycerol kinase (GK) from
a hyperthermophilic archaeon Pyrococcus kodakaraensis
KOD1, was cloned and expressed in Escherichia coli. The
amino acid sequence of this enzyme (Pk-GK) deduced from
the nucleotide sequence showed 57% identity with that of
E.coli GK and 47% identity with that of human GK.
Pk-GK, which has a molecular weight of 55 902 (497 amino
acid residues), was purified from E.coli and characterized.
Despite the high sequence similarity, Pk-GK and E.coli GK
are greatly divergent in structure and function from each
other. Unlike E.coli GK, which exists as a tetramer, Pk-GK
exists as a dimer. The preferred divalent cation for Pk-GK
is Co21, instead of Mg21. The optimum pH and temperature
for Pk-GK activity are 8.0 and 80°C, respectively. Pk-GK
can utilize other nucleoside triphosphates than ATP as a
phosphoryl donor. It is fairly resistant to an allosteric
inhibitor of E.coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the Km
value of the enzyme is 15.4 µM for ATP and 111 µM for
glycerol and its kcat value is 940 s–1. The enzyme was shown
to be fairly resistant to irreversible heat inactivation and
still retained 50% of its enzymatic activity even after
heating at 100°C for 30 min. Construction of a model for
the three-dimensional structure of the enzyme suggests that
the formation of extensive ion-pair networks is responsible
for the high stability of this enzyme.
Keywords: glycerol kinase/hyperthermophilic archaeon/ionpair networks/molecular cloning/subunit structure
Introduction
Glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase, GK) catalyzes Mg,ATP-dependent phosphorylation of
glycerol to produce glycerol-3-phosphate (Lin, 1976), which is
an important metabolic intermediate for both energy production
(glycolysis) and phospholipid biosynthetic pathways. The
enzyme is ubiquitously present in various organisms from
bacteria to human and a number of the genes encoding GK
have been cloned from them and sequenced. They include the
glpK genes from Escherichia coli (Pettigrew et al., 1988),
Bacillus subtilis (Holmberg et al., 1990), Pseudomonas aeruginosa (Schweizer et al., 1997) and Enterococcus casseliflavus
(Charrier et al., 1997), the GUT1 gene from Saccharomyces
© Oxford University Press
cerevisiae (Pavlik et al., 1993) and the Xp gene from humans
(Sargent et al., 1994). In addition, complete determination of
the whole genome sequences of several organisms provides
information on the amino acid sequences of GKs, such as that
from Archaeoglobus fulgidus, which has been deposited in
GenBank with accession number AE001044. Comparison of
these sequences suggests that the amino acid sequence of GK
is relatively well conserved among various GKs. For example,
the identity in the amino acid sequence between E.coli GK
and human GK is 51%. This is probably because GK is a key
enzyme in glycerol metabolism and therefore the enzyme
cannot have become structurally and functionally greatly
divergent during evolution.
Among various GKs, E.coli GK has been most extensively
studied with respect to its functions and structures. This
enzyme acts in the rate-limiting step in the glycerol utilization
pathway and its catalytic activity is closely regulated by
two allosteric inhibitors, IIIglc [EIIAGlc according to a new
nomenclature proposed by Saier and Reizer (1992)] of the
phosphotransferase system (PTS) and fructose-1,6-bisphosphate (FBP) (Thorner and Paulus, 1973; de Riel and Paulus,
1978a,b; Novotny et al., 1985; Liu et al., 1994). The enzyme
is a tetrameric protein composed of identical subunits (Thorner
and Paulus, 1971), but can be dissociated into dimers at low
protein concentrations (de Riel and Paulus, 1978b). The
interaction between the enzyme and one of the substrates,
ATP, showed negative cooperativity, whereas the interaction
between the enzyme and another substrate, glycerol, followed
Michaelis–Menten kinetics (Thorner and Paulus, 1973;
Novotny et al., 1985; Pettigrew et al., 1990). Kinetic and
substrate binding studies have suggested that the dimer is the
enzyme form in the assay and its subunits display different
ATP binding affinities (Pettigrew et al., 1990). The crystal
structure of the enzyme in a complex with the unphosphorylated
form of EIIAGlc, glycerol and ADP (Hurley et al., 1993), and
also that in which glycerol is replaced by glycerol 3-phosphate
(Feese et al., 1994), were determined at 2.6 Å resolution.
According to these structures, unphosphorylated EIIAGlc binds
far from the active site of GK, in which glycerol and ADP
binds, through the formation of an intermolecular Zn(II)
binding site. Thus, it has been postulated that long-range
conformational changes mediate the inhibition of GK by
EIIAGlc. Likewise, a dimer–tetramer assembly reaction has
been postulated to mediate the inhibition of GK by FBP
(de Riel and Paulus, 1978a).
It is well known that all organisms can be evolutionarily
grouped into three classes, eukarya, bacteria and archaea
(Woese and Fox, 1977; Woese et al., 1990). Among them,
archaea, especially hyperthermophilic ones, are expected to
retain traces of early life forms and to produce enzymes which
may represent prototypes in the same protein family, because
they can grow in extreme environments which are probably
similar to that of the primitive earth. Therefore, it would be
informative to analyze the structures and functions of GKs
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Y.Koga et al.
from hyperthermophilic archaea, which may be different from
those of GKs from bacteria and eukarya. Consequently, these
studies may serve to elucidate the adaptation mechanisms of
proteins to high temperature and the molecular evolution of
the enzyme. However, none of the GK enzymes has been
isolated from archaea. Therefore, we decided to characterize
GK from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.
Pyrococcus kodakaraensis KOD1, which has previously
been designated Pyrococcus sp. KOD1, was isolated from a
solfatara at a wharf on Kodakara Island, Kagoshima, Japan
(Morikawa et al., 1994). The growth temperature of this strain
ranged from 65 to 100°C and the optimal temperature is 95°C.
Here we report that GK from P.kodakaraensis KOD1 showed
distinctive structural and enzymatic characteristics as compared
with those from mesophilic sources. Like other enzymes from
this strain, it is more stable than the bacterial enzymes and
shows broad metal ion and NTP specificities. In addition,
unlike E.coli GK, it exists in a dimeric form, is not sensitive
to FBP inhibition and does not show negative cooperativity
for ATP binding. We will discuss on the structure–function
relationships of this enzyme, based on a model for the threedimensional structure.
Materials and methods
Cells and plasmids
Pyrococcus kodakaraensis KOD1 was isolated from a solfataric
hot spring at a wharf on Kodakara Island, Kagoshima, Japan
(Morikawa et al., 1994). Escherichia coli strain JM109 [recA1,
endA1, gyrA96, thi, hsdR17, SupE44, relA1, λ–, ∆(lac-ProAB)/
F9, traD36, ProA1B1, lacIqZ∆M15] and plasmid pUC19 for
DNA manipulations were obtained from Toyobo. E.coli strain
BL21(DE3) [F–, ompT, hsdSB(rB–mB–), gal, dcm(DE3)] and
plasmid pET-8c for gene expressions were obtained from
Novagen.
Materials
Restriction enzymes, DNA polymerase and T4 DNA ligase
were obtained from Takara Shuzo. Glycerol-3-phosphate oxidase (GPO) and peroxidase (PO) were kindly provided by
Toyobo. [1,3-14C]glycerol (100 mCi/mmol) was purchased
from ICN.
Cloning of the Pk-glpK gene
A portion of the Pk-glpK gene was first amplified by PCR
with a combination of forward (59-TGCTGTTTGGTACGG39) and reverse (59-AGCCAGTGCCATAGGTGTTCTTCG39) primers. The genomic DNA, which was prepared from a
sarkosyl lysate of P.kodakaraensis KOD1 cells as described
previously (Imanaka et al., 1981), was used as a template. The
resulting 318-base pair (bp) DNA fragment was used as a
probe for Southern blotting and colony hybridization to clone
the entire Pk-glpK gene.
PCR was performed in 30 cycles with a Perkin-Elmer
thermal cycler (GeneAmp PCR System 2400) using Vent
polymerase (New England Biolabs), as described previously
(Kim et al., 1995). All DNA oligomers were synthesized by
Sawady Technology. General DNA manipulations were carried
out as described previously (Sambrook et al., 1989). The
nucleotide sequence was determined by the dideoxy-chain
termination method (Sanger et al., 1977) with an ABI PRISM
310 genetic analyzer (Perkin-Elmer).
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Overexpression and purification of Pk-GK
The Pk-glpK gene was amplified by PCR with a combination
of forward (59-ATATACCATGGAAAAGTTCGTTCTTTC39) and reverse (59-AATGGATCCATATCAATTTGATTTTGCACT-39) primers, where the underlines represent the NcoI
and BamHI sites, respectively, and the ATG codon for the
initiation of the translation and the sequence complementary
to the termination codon TGA are shown in italics. The
resultant 1.5-kb DNA fragment was digested with NcoI and
BamHI and cloned into the NcoI–BamHI site of plasmid pET8c to create plasmid pET-pkgk. An overproducing strain for
Pk-GK was constructed by transforming E.coli BL21(DE3)
with this plasmid.
For the overproduction of Pk-GK, an E.coli BL21(DE3)
transformant with pET-pkgk was grown at 37°C in NZCYM
medium (Novagen) containing 50 µg/ml ampicillin. When the
absorbance at 600 nm of the culture reached ~0.6, 1 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the
culture medium and cultivation was continued for an additional
8 h. Cells were then harvested by centrifugation at 6000 g for
10 min and subjected to the purification procedures.
Cells were suspended in 50 mM sodium phosphate buffer
(pH 7.5), disrupted by sonication with a Model 450 sonifier
(Branson Ultrasonic) and centrifuged at 15 000 g for 30 min.
The supernatant was heated at 90°C for 30 min and then
applied to a column of MonoQ (Pharmacia) equilibrated with
the same buffer. The enzyme eluted from the column as a
single peak at an NaCl concentration of ~0.5 M by linearly
increasing the NaCl concentration from 0 to 1.0 M in the same
buffer. All purification procedures, except for heat treatment,
were carried out at 4°C. The production level and the purity
of the enzyme were analyzed by SDS–PAGE (Laemmli, 1970).
Determination of enzymatic activity
The enzymatic activity of Pk-GK was determined in 800 µl
of 50 mM glycine–NaOH (pH 8.0) containing 2 mM CoCl2,
4.6 mM glycerol and 3.5 mM ATP at 80°C, unless stated
otherwise. After incubation for 5 min, the reaction was
terminated by the addition of 16 µl of 0.5 M EDTA and the
reaction mixture was kept on ice for a few minutes. The
amount of glycerol-3-phosphate (G-3-P) in the reaction mixture
was then determined colorimetrically at 37°C by coupled
enzyme reactions catalyzed by glycerol-3-phosphate oxidase
(GPO) and peroxidase (PO) as described previously (Bergmeyer et al., 1961). One unit of enzymatic activity was defined
as the amount of enzyme producing 1 µmol of glycerol-3phosphate in 1 min. The specific activity was defined as the
enzymatic activity per milligram of protein.
For determination of the kinetic parameters the assay solution
contained, in a volume of 100 µl, 50 mM Tris–HCl (pH
7.5), 2 mM CoCl2, 2 mM or appropriate concentration of
[14C]glycerol, 2 mM or appropriate concentration of ATP and
0.06 µg of Pk-GK. After incubation for 5 min at 80°C, the
reaction was terminated by the addition of 2 µl of 0.5 M
EDTA. An aliquot of the reaction mixture was then applied to
a DEAE-cellulose filter-paper (diameter 2.4 cm) (Toyo Roshi).
After washing the filter-paper with 0.1 M glycerol, the amount
of [14C]G-3-P bound to the paper was determined using
Instant Imager (Packard). The production of G-3-P followed
Michaelis–Menten kinetics and the kinetic parameters, Km and
kcat, were determined from the Lineweaver–Burk plot.
Protein concentration was determined from the UV absorption at 280 nm by using an A2800.1% value of 2.2 (molar
Glycerol kinase from a hyperthermophilic archaeon
absoptivity 1.233105 l/mol.cm at 280 nm). This value was
determined by measuring the amount of the protein by amino
acid analysis
Biochemical characterization
The molecular weight of Pk-GK was estimated by gel filtration
on a column of HiLoad Superdex 200 (Pharmacia) equilibrated
with 10 mM Tris–HCl (pH 8.0) containing 150 mM NaCl.
Standard molecular marker (Bio-Rad) was applied to the
column for calibration of molecular weight. The N-terminal
amino acid sequence of the enzyme was determined with a
Procise automated sequencer (Perkin-Elmer Model 491).
Amino acid analysis was carried out with a Hitachi L-8500
automatic amino acid analyzer. Samples were hydrolyzed at
110°C for 24 h with 6 M HCl containing 0.2% (v/v) phenol.
Circular dichroism (CD) measurement
CD spectra were measured on a J-720W spectropolarimeter
(Japan Spectroscopic). Far-UV (200–260 nm) and near-UV
(250–320 nm) CD spectra were obtained at 30°C by using a
solution containing enzyme at 0.13 and 0.25 mg/ml in 50 mM
sodium phosphate buffer (pH 7.9) containing 0.5 M NaCl in
a cell with an optical path of 2 and 10 mm, respectively. The
mean residue ellipticity, [θ], which has the units of ° cm2/
dmol, was calculated by using an average amino acid molecular
weight of 110.
Construction of a three-dimensional model of Pk-GK
A tertiary model of Pk-GK was constructed by homology
modeling using the following programs: a loop search method
for the backbone structure (Nakamura et al., 1991), a dead-end
elimination method for the side-chain conformation (Tanimura
et al., 1994) and conformation energy minimization for the
structure refinement (Morikami et al., 1992), using the AMBER
force field (Weiner et al., 1986).
Results
Cloning and sequencing of the Pk-glpK gene
On the assumption that conserved amino acid sequences in
various glycerol kinases (GKs) are also conserved in Pk-GK
and the nucleotide sequence of the Pk-glpK gene is similar to
that of the E.coli glpK gene in the regions where the amino
acid sequences encoded are conserved, various DNA oligomers
(15–25 bases) with sequences identical with those in the E.coli
glpK gene were synthesized and used as a primer to amplify
a portion of the Pk-glpK gene by PCR. Among various
combinations of forward and reverse primers examined, a
combination of the forward primer (15 bases) encompassing
the sequences coding for amino acids 161–166 of E.coli GK
and the reverse primer (24 bases) encompassing the sequences
coding for amino acids 261–269 of E.coli GK was shown to
be effective in amplifying the 318-bp DNA fragment from the
KOD1 genome. Determination of the DNA sequence indicated
that this DNA fragment is a portion of the Pk-glpK gene,
which encodes the amino acid sequence of Pk-GK from
residues 157 to 263. By using this DNA fragment as a probe
for Southern hybridization, the 4-kb BamHI fragment or 2-kb
HindIII fragment was shown to contain this 318-bp DNA
fragment (data not shown). Determination of the DNA sequence
indicated that the 4-kb BamHI fragment contains the entire
Pk-glpK gene, whereas the 2-kb HindIII fragment contains a
part of it, in which the 39-terminal region is truncated,
because the HindIII site is located within the Pk-glpK gene.
Identification of a strong ribosomal binding signal (59-
AGGTGAT-39), which is complementary to the 39-terminal
sequence of the 16S rRNA from KOD1 (fourth to tenth
residues from the 39-terminus) (Morikawa et al., 1994), at the
59-terminal region of a long uninterrupted reading frame with
~1600 bases allowed us to choose the AUG codon, which is
located six bases downstream from this ribosomal binding
signal, for initiation of translation. The protein from this region
would be composed of 497 amino acid residues with a
calculated molecular weight of 55 902. A possible TATA-like
promoter site (59-TTAAAAA-39, A-box) is located 67 base
pairs upstream from the initiation codon and a possible
transcription termination signal with a tandem repeat of thymine is located ~30 base pairs downstream from the termination
codon. The nucleotide sequence of the Pk-glpK gene, and also
its flanking sequences, are deposited in DDBJ with accession
number AB012099.
Comparison of amino acid sequences
The amino acid sequence of Pk-GK deduced from the nucleotide sequence 1s compared with those of GKs from E.coli,
Archaeoglobus fulgidus and humans in Figure 1. These
sequences were chosen as representatives of those of GKs
from bacteria, archaea and eukaryotes. Among various GK
sequences so far available, the E.coli GK sequence gave the
closest similarity to the Pk-GK sequence with an identity of
57%. The amino acid sequence identities between Archaeoglobus fulgidus GK and Pk-GK and between human GK and
Pk-GK were 36 and 47%, respectively. All of the amino acid
residues, except for Ile313, which have been shown to be
involved in the bindings with glycerol, ADP and Mg21 for
E.coli GK (Hurley et al., 1993) are fully conserved in the PkGK sequence. They are Arg82, Glu83, Trp101, Tyr132, Asp239
and Phe264 for glycerol binding, Asp9 and Asp239 for catalytic
function and probably for divalent cation binding and Arg16,
Gly260, Thr261, Gly303, Gln307, Ala319, Leu374, Ile377,
Gly402, Ala403 and Asn406 for ADP binding. Ile313 of E.coli
GK, which contributes to form a hydrophobic pocket for the
binding of adenine base, is replaced by Val (Val306) in the
Pk-GK sequence.
Overproduction and purification
In order to facilitate the preparation of Pk-GK in an amount
sufficient for biochemical characterizations, we constructed
plasmid pET-pkgk, in which transcription of the Pk-glpK gene
is initiated by the T7 promoter. This plasmid was used to
transform E.coli BL21(DE3) to ampicillin resistance. Induction
of the gene by the addition of IPTG caused accumulation of
the enzyme as the most abundant protein in the cells (Figure
2). The production level of the enzyme was estimated to be
~40 mg/l culture by SDS–PAGE as shown in Figure 2. Because
the amount of the enzyme in the soluble fraction obtained
after sonication lysis (crude lysate) was comparable to that in
the whole cell extract, the enzyme should be accumulated in
cells in a soluble form (data not shown). From the crude
lysate, the enzyme was purified in only two steps with a yield
of 30–40% (Table I). The purified enzyme gave a single band
on SDS–PAGE with a molecular weight of 55 000 (Figure 2).
This value was closely comparable to that calculated from its
amino acid sequence.
Biochemical properties
To confirm the amino acid sequence of Pk-GK predicted from
the DNA sequence, the NH2-terminal amino acid sequence
and amino acid composition of the protein were determined.
1221
Y.Koga et al.
Fig. 2. Comparison of the purity of Pk-GK by SDS–PAGE. Samples were
subjected to electrophoresis on a 10% polyacrylamide gel in the presence of
SDS. After electrophoresis, the gel was stained with Coomassie Brilliant
Blue. Lane 1, a low molecular weight marker kit (Pharmacia LKB
Biotechnology) containing phosphorylase b (94K), bovine serum albumin
(67K), ovalbumin (43K), carbonic anhydrase (30K), trypsin inhibitor (20K)
and α-lactalbumin (14K); lane 2, crude extract from E.coli BL21(DE3)
transformant with pET-pkgk; lane 3, purified Pk-GK.
Table I. Purification of recombinant Pk-GK from a 1 l culture of E.coli
cells
Fraction
Total protein Total activity Specific activity Yield (%)
(mg)
(units)
(units/mg)
Crude extract
162
Heat treatment
36
Mono Q fraction
9.2
Fig. 1. The alignment of the Pk-GK, E.coli GK, A. fulgidus GK and human
GK sequences. The amino acid residues of GKs from E.coli, A.fulgidus and
humans, which are identical with those of Pk-GK, are indicated by a dash
(–). For human GK, the sequence from Ala7 to Asp523 is shown. The
ranges of the 16 α-helices and the 25 β-strands of E.coli GK are shown
above the sequences, according to the crystal structure determined by
Hurley et al. (1993). Numbers above the sequences indicate the position of
the residue in Pk-GK, which starts from the initiator methionine. Numbers
along the sequences indicate the position of the amino acid residue listed in
the end of each line for GKs from E.coli, A.fulgidus and humans. The
amino acid residues that are possibly involved in the catalytic function and
the substrate-binding of Pk-GK are shown in bold face. The regions in the
E.coli GK sequence, which are involved in the subunit-subunit interactions
(Hurley et al., 1993), are shaded. O–X, O–Y and O–Z in parentheses
represent the three types of the subunit–subunit interactions. The sequences
have been deposited in Swiss-Prot with code numbers P08859 for E.coli GK
(E. co) and P32189 for human GK (Hum), in EMBL/GenBank with
accession number AE001044 for A.fulgidus GK (A. fu) and in DDBJ with
accession number AB012099 for Pk-GK.
The sequence of the NH2-terminal 10 amino acid residues was
MEKFVLSLDE, which was identical with that predicted from
the DNA sequence. In addition, there is good agreement
between the amino acid composition predicted from the DNA
1222
13040
7160
4140
80
199
450
100
54.9
31.7
Fig. 3. Far- and near-UV CD spectra of Pk-GK. The CD spectra were
measured in 50 mM sodium phosphate buffer (pH 7.9) containing 0.5 M
NaCl at 30°C as described under Materials and methods. The mean residue
ellipticity, [θ], is given in units of °·cm2/dmol.
sequence and that determined experimentally (data not shown).
The molecular weight of the enzyme in its native state was
estimated to be 125 000 by gel filtration chromatography (data
not shown). This result indicates that the enzyme exists in a
dimeric form. The far-UV CD spectrum of the protein exhibited
a trough with a minimum [θ] value of –13 600 at 210 nm,
which is accompanied by a shoulder with a [θ] value of
–12 500 at 220 nm (Figure 3a). The near-UV CD spectrum of
the protein exhibited two broad peaks with [θ] values of 700
at 260 and 275 nm and a trough with a [θ] value of –400 at
Glycerol kinase from a hyperthermophilic archaeon
Fig. 4. Temperature and pH dependence of Pk-GK activity. (a) The
enzymatic activity was determined in 50 mM glycine–NaOH (pH 8.0)
containing 2 mM CoCl2, 4.6 mM glycerol and 3.5 mM ATP at the
temperatures indicated. The enzymatic activities relative to that determined
at 80°C are shown. (b) The enzymatic activity was determined in 50 mM
Tris–HCl (pH 6.5–8.0) (s) or 50 mM glycine–NaOH (pH 7.5–10.0) (d)
containing 2 mM CoCl2, 4.6 mM glycerol and 3.5 mM ATP at 80°C. The
enzymatic activities relative to that determined in 50 mM Tris–HCl (pH 8.0)
are shown. In both cases, the amount of glycerol-3-phosphate was
determined colorimetrically at 37°C as described previously (Bergmeyer
et al., 1961).
Table II. Effect of divalent cations on Pk-GK activity
Compound
Concentration (mM)
None
MgCl2
CaCl2
ZnCl2
CoCl2
FeCl2
CuSO4
MnCl2
NiCl2
SrCl2
BaCl2
–
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Relative activity (%)
0.6
49.3
4.0
99.3
100
,0.1
4.0
78.0
57.3
0.3
0.2
The enzymatic activity was determined in 50 mM glycine–NaOH (pH 8.0)
containing various divalent cations, 4.6 mM glycerol and 3.5 mM ATP at
80°C. The amount of glycerol-3-phosphate was determined colorimetrically
at 37°C as described previously (Bergmeyer et al., 1961). The relative
activities were calculated by dividing the enzymatic activities determined in
the presence of the various divalent cations by that determined in the
presence of the Co21 ion.
297 nm (Figure 3b). The helical content of the protein was
calculated as 32.5% by the method of Wu et al. (1981).
Enzymatic activity
Analysis of the temperature and pH dependence of the Pk-GK
activity indicated that the optimal values for activity were
80°C (Figure 4a) and pH 8.0 (Figure 4b). The enzyme exhibited
little activity in the absence of any divalent cation, indicating
that the enzyme requires divalent cations for activity. To
identify the preferred metal ion for activity, the enzymatic
activity was determined at 80°C and pH 8.0 in the presence
of salts of various divalent cations, such as CaCl2, ZnCl2,
CoCl2, FeCl2, CuSO4, MnCl2, NiCl2, SrCl2 and BaCl2. The
results are summarized in Table II. The enzyme exhibited the
highest activity in the presence of Co21 or Zn21, which was
nearly twice as high as that determined in the presence of
Mg21. It also exhibited relatively high activity in the presence
of Mn21 and Ni21, but exhibited little or poor activity in the
presence of other metal ions. When the concentration of the
metal ion was varied from 0.5 to 4 mM, the enzyme exhibited
the highest activity in the presence of 1–2 mM metal ion,
Fig. 5. Stability against irreversible heat inactivation. The solution
containing Pk-GK at 2.7 µg/ml in 50 mM glycine–NaOH (pH 8.0) was
incubated at 90°C (d) or 100°C (s). At appropriate intervals, an aliquot of
the enzyme solution was withdrawn and analyzed for remaining activity.
The remaining activity is shown as a function of incubation time.
regardless of the species of metal ion. Thus, the enzyme
exhibited the highest specific activity of 450 units/mg (turnover
number 840 s–1) at 80°C and pH 8.0 in the presence of 2 mM
CoCl2. Because the enzymatic activity determined in the
presence of 0.5 M NaCl or KCl was similar to that determined
in the absence of salt, the enzyme neither requires salt for
activity nor is sensitive to salt.
In addition to the divalent cation specificity, the nucleoside
triphosphate specificity of the enzyme was analyzed by replacing ATP in the assay buffer with other nucleoside triphosphates, such as GTP, CTP, UTP and ITP. The enzymatic
activities determined in the presence of 3.3 mM GTP, CTP,
UTP and ITP were 3.4, 15.9, 12.1 and 14.2% of that determined
in the presence of 3.5 mM ATP, respectively. These results
indicate that the enzyme can use various nucleoside triphosphates as a phosphoryl group donor. Since it has been proposed
that E.coli GK in a tetrameric form is non-competitively
inhibited by FBP, but the enzyme in a dimeric form is not (de
Riel and Paulus, 1978a; Liu et al., 1994), Pk-GK was also
tested for inhibition by FBP. The enzyme retained 71% of the
activity even in the presence of 20 mM FBP, suggesting that
the Pk-GK activity is not sensitive to FBP.
ATP and glycerol are the substrates of Pk-GK for the
production of G-3-P. Therefore, the kinetic parameters of PkGK were determined in the presence of an excess amount of
either of these substrates. For example, to determine the Km
and kcat values for glycerol, the concentration of ATP was
fixed at 2 mM and the concentration of glycerol was varied.
The Km values for ATP and glycerol were determined as 15.4
and 111 µmol, respectively. The kcat value was determined as
940 s–1.
Heat inactivation
The stability of Pk-GK against irreversible heat inactivation
was analyzed by incubating the enzyme solution (2.7 µg/ml)
in 50 mM sodium phosphate buffer (pH 7.5) at 90 or 100°C.
At appropriate intervals, an aliquot of the enzyme solution
was withdrawn, chilled on ice and analyzed for remaining
activity. As shown in Figure 5, the enzyme is fairly resistant
to thermal denaturation at 90°C. The enzyme gradually lost
activity at 100°C with a half-life of ~30 min.
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Y.Koga et al.
Discussion
Amino acid sequence of recombinant Pk-GK
The NH2-terminal methionine residue is not post-translationally
removed from recombinant Pk-GK, but is removed from E.coli
GK (Pettigrew et al., 1988). It has been proposed that the
second amino acid residue determines whether the NH2terminal methionine residue is removed or not (Tsunasawa
et al., 1985). According to this proposal, the NH2-terminal
methionine residue is removed when the second amino acid
is Ala, Ser, Gly, Pro, Thr or Val, whereas it is not removed
when the second amino acid is Arg, Asn, Asp, Gln, Glu, Ile,
Leu or Lys. The observation that the NH2-terminal methionine
residue is not removed from Pk-GK but is removed from
E.coli GK supports this proposal, because the second amino
acid is Glu for Pk-GK and Thr for E.coli GK. However, it
remains to be determined whether natural Pk-GK has Met at
its NH2-terminus as well.
Enzymatic activity of Pk-GK
The Km value of E.coli GK for glycerol has been reported to
be 1.3 µM (Hayashi and Lin, 1967), 7 µM (Novotny et al.,
1985) or 10 µM (Thorner and Paulus, 1973). Because the
interaction of this enzyme with ATP shows negative cooperativity, two Km values of the enzyme for low and high concentrations of ATP have been reported to be 60 and 900 µM
(Novotny et al., 1985) or 80–100 and 400–500 µM (Thorner
and Paulus, 1973), respectively. The Km value of 4 mM, which
was the only one reported for ATP by Hayashi and Lin (1967),
may represent that for high concentrations of ATP. Interestingly,
Pk-GK did not show negative cooperativity for the interaction
with ATP. The Km value of this enzyme for ATP (15.4 µmol)
was much lower than those of E.coli GK, suggesting that the
binding affinity of ATP to Pk-GK is much higher than that to
E.coli GK. In contrast, the Km value of this enzyme for glycerol
(111 µmol) was much higher than that of E.coli GK, suggesting
that the binding affinity of glycerol to Pk-GK is much lower
than that to E.coli GK. A difference in the subunit structure
or a slight difference in the conformation of the active site
may be responsible for the alteration in the binding affinities
of these substrates to the enzyme.
The specific activity of Pk-GK (450 units/mg) was comparable to that of E.coli GK, which has been reported to be 1004
units/mg (Thorner and Paulus, 1973), indicating that the
catalytic efficiencies of Pk-GK at 80°C and E.coli GK at mild
temperature are similar to each other. Because local instability
usually makes the active site conformationally flexible and
thereby makes the enzyme functional (Meiering et al., 1992;
Imoto et al., 1994; Shoichet et al., 1995; Kanaya et al., 1996),
the conformational flexibility of the active site of Pk-GK
gained at high temperature might be similar to that of E.coli
GK gained at mild temperature. Pk-GK exhibited a very low
enzymatic activity at 40°C (Figure 4a). The active site of
Pk-GK may be conformationally too stable to display sufficient
flexibility at mild temperature.
E.coli GK can use only ATP as a phosphoryl group donor
(Hayashi and Lin, 1967). In contrast, Pk-GK can utilize other
trinucleoside phosphates than ATP as a phosphoryl group
donor, although the enzymatic activities determined in their
presence are considerably lower than that determined in
the presence of ATP. The amino acid residues that form a
hydrophobic pocket for the binding of adenine base in E.coli
GK are fully conserved in Pk-GK, except for Ile313, which is
replaced by Val306 in Pk-GK. Therefore, this amino acid
1224
Fig. 6. A tertiary model of Pk-GK monomer and the X-ray crystal structure
of E.coli GK monomer. A model of Pk-GK monomer (top) and the crystal
structure of E.coli GK monomer (PDB code 1GLB) (Hurley et al., 1993)
(bottom) are shown. White pipe models are the backbone structures. The
blue and red lines are the basic and acidic side-chains, respectively, which
are involved in the ion pairs as indicated in Tables III and IV. The orange
lines indicate the ion pairs that are only observed in Pk-GK or E.coli GK in
Table III. The green lines indicate the ion-pair networks in Table IV. The
bound ADP and glycerol at the active site are also shown by yellow spacefilling models. The side-chains of Val306 in Pk-GK (top) and Ile313 in
E.coli GK (bottom) are also indicated by green space-filling models. N and
C represent the NH2 and COOH termini of the molecules. The figure was
drawn with the program Insight II (Molecular Simulations).
substitution may be related to a relatively poor nucleoside
triphosphate specificity of Pk-GK. In addition to Pk-GK, GKs
from rat liver (Bublitz and Kennedy, 1954) and Candida
mycoderma (Bergmeyer et al., 1961) have been shown to be
active with nucleoside triphosphates other than ATP. However,
the sequence information is not available for either of these
enzymes.
All GKs so far examined, except for GK from Thermus
flavus, which exhibits activity in the presence of 1 mM EDTA
(Huang et al., 1997), require divalent cations for activity.
The preferred metal ion for E.coli GK activity is Mg21 and
only the Mn21 ion can be substituted for it (Hayashi and Lin,
1967). On the other hand, Pk-GK is active with Co21, Zn21,
Glycerol kinase from a hyperthermophilic archaeon
Table III. Intramolecular ion pairs (ø4.0 Å)a
Conservedb
Pk-GK
E.coli GK
Arg17–Asp10
Arg83–Glu303
Arg106–Asp133
Arg107–Glu46
Lys116–Glu113
Lys142–Asp146
Arg152–Asp146
Arg152–Asp208
Arg154–Glu90
Arg154–Glu159
Arg188–Glu84
Arg188–Glu303
Arg218–Glu216
Arg218–Glu222
Arg317–Asp318
Arg378–Glu382
Arg432–Glu467
Lys487–Asp390
Arg22–Glu23
Arg22–Glu450
Arg67–Glu69
Lys122–Glu123
Lys124–Asp195
Lys124–Asp197
Lys139–Glu112
Lys154–Glu150
Arg169–Glu49
Lys190–Glu123
Lys191–Asp193
His318–Glu321
Lys328–Glu367
Arg382–Glu471
Lys395–Glu396
Arg419–Glu389
Lys420–Glu396
Arg423–Asp407
Arg449–Asp447
Arg459–Asp407
Lys464–Glu462
Arg470–Glu471
Lys482–Glu479
Arg483–Glu479
Arg33–Glu62
His114–Glu110
Arg125–Glu121
Arg156–Glu153
Arg211–Asp198
Arg361–Glu497
Arg389–Glu393
Arg407–Glu431
Arg482–Glu478
aThe
ion pairs formed in Pk-GK are hypothetical, because the distance
between two ionic residues was calculated according to a model of the
three-dimensional structure of the enzyme. The conserved ion pairs and also
the ion pairs that are formed only in Pk-GK or E.coli GK are listed.
bThe ion pairs formed in E.coli GK are listed as representatives of those
formed in common. All of these residues, except Arg154 and Glu90, which
are replaced by Lys151 and Asp89, respectively, in Pk-GK, are conserved in
Pk-GK.
Table IV. Intramolecular ion-pair networks (ø4.0 Å)
Pk-GK
E.coli GK
Arg82–Glu296–Arg183–Glu83
Arg83–Glu303–Arg188–Glu84
Glu112–Lys139–Asp143–Arg149–Asp203 Lys142–Asp146–Arg152–Asp208
Asp89–Lys151–Glu156
Glu90–Arg154–Glu159
Glu23–Arg22–Glu450
Lys122–Glu123–Lys190
Arg382–Glu471–Arg470
Lys395–Glu396–Lys420
Glu458–Arg423–Asp407–Arg459
Lys482–Glu479–Arg483
The ion-pair networks that are formed in Pk-GK or E.coli GK are listed.
Fig. 7. Schematic representation for the tetrameric structure of E.coli GK.
The subunit–subunit interactions in the tetrameric structure of E.coli GK are
shown schematically. The subunits X, Y and Z are related to the subunit O
by a 2-fold rotation about the X, Y and Z axes, respectively. The active site
of each subunit is designated by a solid square. The dimeric structure (O–Y
dimer) shaded is expected to represent that of Pk-GK.
Mg21, Ni21 or Mn21. Because the proteins isolated from
P. kodakaraensis KOD1 sometimes show a wide preference
for metal ions (Rashid et al., 1997a,b), it may be a characteristic
of the proteins from hyperthermophilic archaea.
Pk-GK was shown to be rather resistant to the FBP inhibition.
Because Pk-GK seems to act as a dimer, this result is consistent
with the previous reports that FBP binds and inhibits only the
tetrameric form of E.coli GK (de Riel and Paulus, 1978a; Liu
et al., 1994). We have not analyzed whether the Pk-GK activity
is inhibited by another allosteric inhibitor, EIIAGlc, because
neither the gene encoding EIIAGlc nor the EIIAGlc protein from
hyperthermophilic archaea is available. We could not identify
the gene encoding a homologue of EIIAGlc in the DNA
sequence of the Archaeoglobus fulgidus genome.
Stability of Pk-GK
Because Pk-GK seems to be conformationally fairly stable
and functionally well adapted to a hyperthermal environment
and because it shows a high sequence similarity to E.coli GK,
Pk-GK and E.coli GK may be an ideal protein pair to analyze
the mechanism by which proteins from hyperthermophiles
become more stable than the mesophilic counterparts. Comparison of the crystal structures of glutamate dehydrogenase (Yip
and Rice, 1995; Knapp et al., 1997) and indole-3-glycerol
phosphate synthase (Hennig et al., 1995) from hyperthermophilic archaea with those from mesophilic counterparts have
suggested that an increase in the number of ion pairs (ion-pair
networks) and a decrease in the volume of cavities are
responsible for the hyperstability of these hyperthermophilic
proteins. It has been noted that proteins from the thermophilic
sources have extra ion-pair networks on the protein surface
(Perutz, 1978; Nakamura, 1996). To examine whether the
number of ion pairs or ion-pair networks increased in Pk-GK
as compared with that in E.coli GK, a model for the threedimensional structure of Pk-GK was constructed and compared
with that of E.coli GK (Figure 6). Because Pk-GK shows a
high sequence similarity with E.coli GK, its overall backbone
structure is expected to be nearly identical with that of E.coli
GK. In fact, the helical content of Pk-GK (32.5%) estimated
from the far-UV CD spectrum was comparable to that of E.coli
GK (36.3%) determined from crystallographic studies (Hurley
et al., 1993). Apparently, the number of the charged residues
involved in the formation of ion pairs and ion-pair networks
at the surface of the Pk-GK molecule is higher than that in
the E.coli GK molecule (Figure 6).
All possible intramolecular ion pairs and ion-pair networks
formed only in Pk-GK or E.coli GK, which were estimated
by the method of Barlow and Thornton (1983), are listed in
Tables III and IV, respectively. Twenty-four ion pairs and nine
ion-pair networks are formed only in Pk-GK, whereas only
nine ion pairs and three ion-pair networks are formed only in
E.coli GK. Eighteen ion pairs are formed in common in these
proteins. Thus, an increase in the number of ion pairs and ionpair networks may contribute to the increase in the protein
stability of Pk-GK. Estimation of the number of the ion
pairs formed in GKs from A. fulgidus, which is an another
hyperthermophilic archeon and humans also supports this
prediction. The number of the ion pairs in thermophilic GK
from T.flavus (Huang et al., 1997) cannot be estimated, because
the sequence information is not available for this protein. We
are now trying to analyze the conformational stability of these
proteins thermodynamically and the effect of the introduction
or elimination of ion pairs (ion-pair networks) on the protein
stability.
1225
Y.Koga et al.
Subunit structure of Pk-GK
Gel filtration experiments on agarose beads have previously
shown that E.coli GK exists in an equilibrium between a dimer
and a tetramer at concentrations lower than ~0.01 mg/ml,
but exists in a tetramer at concentrations of more than ~0.1
mg/ml (de Riel and Paulus, 1978b). In contrast, Pk-GK exists
as a dimer at a concentration of 0.5 mg/ml. These results
suggest that interactions or forces which are required for the
assembly of the tetramer from the dimer are reduced in the
Pk-GK structure. The crystal structure of E.coli GK (Hurley
et al., 1993), which contains tetramers of GK, is shown
schematically in Figure 7. There are three types of intersubunit
interactions in this GK structure, namely the interactions
between O and Y subunits (O–Y interface), O and X subunits
(O–X interface) and O and Z subunits (O–Z interface). Because
the solvent-accessible surface area of each monomer is most
extensively buried by the O–Y interaction and because the
side chain of Arg369 (Arg362 in Pk-GK) of the Y subunit
penetrates deeply into the O subunit, it has been proposed that
the dimer–tetramer equilibrium involves dissociation of the
tetramer into O–Y dimers (Hurley et al., 1993).
Comparison of the amino acid sequences of Pk-GK and
E.coli GK (Figure 1) indicates that the residues involved in
the O–Y interface (residues 341–347 and 361–367 for E.coli
GK) are well conserved. In contrast, the residues involved in
the O–X interface (residues 49–68 for E.coli GK) and those
involved in the O–Z interface (residues 321–333 for E.coli
GK) are not well conserved. Therefore, it seems likely that
Pk-GK assumes the O–Y dimer form. The observation that
the mutation of Ala65 to Thr or Asp72 to Asn in E.coli
GK shifts the dimer–tetramer equilibrium toward the dimer
supports this prediction (Liu et al., 1994). In Pk-GK, these
residues are replaced by Gln (Gln64) and Asn (Asn71),
respectively.
The hydropathy profiles of Pk-GK and E.coli GK were
determined by the method of Kyte and Doolittle (1982) (data
not shown). The striking similarity in these profiles suggests
that hydrophobic interactions do not determine the subunit
structures of the enzyme.
Role of Pk-GK
It has been shown that the glycerophosphate backbone of
archaeal phospholipids is glycerol-1-phosphate (G-1-P),
instead of G-3-P (Koga et al., 1993). This raises the question
as to whether Pk-GK catalyzes the production of G-1-P as
well. However, the enzymatic activity of Pk-GK determined
by the calorimetric method, in which only the amount of G3-P can be determined, was nearly identical with that determined by the filter method, in which the amount of the 14Clabeled glyceromonophosphate (G-1-P and G-3-P) can be
determined by measuring the radioactivity bound to a DEAEcellulose filter (data not shown). If Pk-GK produced both G1-P and G-3-P, the enzymatic activity determined by the
calorimetric method would be lower than that determined by
the filter method. Therefore, it is clear that a major product of
Pk-GK is G-3-P. Because Pk-GK may not catalyze the production of G-1-P, Pk-GK may be important for energy production,
instead of phospholipid biosynthesis.
The glp regulon
In the chromosome of E.coli, five different operons constitute
a glycerol regulon (glp) (Larson et al., 1992). The glpK gene
forms one of these operons (glpFK) with the glpF gene,
which encodes a membrane protein that facilitates diffusion
1226
of glycerol across a cytoplasmic membrane. This glpFK operon
has also been found in the chromosome of Bacillus subtilis
(Holmberg and Rutberg, 1989). However, determination of the
upstream sequence of Pk-glpK has revealed that the glpF gene
is not located at the upstream region of the Pk-glpK gene
(Y.Koga, personal communication). Instead, the gene encoding
a protein, that is homologous to glycerophosphodiester phosphodiesterase, is located in this region. In the E.coli and
B.subtilis chromosomes, the glpQ gene encoding glycerophosphodiester phosphodiesterase forms an operon (glpTQ) with
the glpT gene encoding glycerol-3-phosphate permease (Larson
et al., 1992; Nilsson et al., 1994). Therefore, the organization of
the genes involved in glycerol metabolism of P.kodakaraensis
KOD1 seems to be different from those of E.coli and B.subtilis.
Analyses for the DNA sequences of the flanking regions of
Pk-glpK are now in progress.
Acknowledgements
We thank Mr N.Yoshiaki of Toyobo Co., Ltd, for providing glycerol-3phosphate oxidase (GPO) and peroxidase (PO) for GK assay. This work was
supported in part by Grant 08455382 from the Ministry of Education, Science
and Culture of Japan.
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Received July 7, 1998; revised August 14, 1998; accepted August 18, 1998
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