JOURNAL OF BACTERIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Oct. 2000, p. 5624–5627Vol. 182, No. 19
Purification and Characterization of glpX-Encoded Fructose
1,6-Bisphosphatase, a New Enzyme of the Glycerol
3-Phosphate Regulon of Escherichia coli
JANET L. DONAHUE, JENNIFER L. BOWNAS, WALTER G. NIEHAUS, AND TIMOTHY J. LARSON*
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
Received 3 February 2000/Accepted 8 July 2000
In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phos-
phate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose
1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was
dimeric, dependent on Mn2?for activity, and exhibited an apparent Kmof 35 ?M for fructose 1,6-bisphosphate.
The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.
Growth of Escherichia coli on glycerol or sn-glycerol 3-phos-
phate (glycerol-P) as the sole carbon source is mediated pri-
marily by the glp regulon (15). The glpFKX operon, one of the
five operons of the regulon, encodes glycerol facilitator (glpF),
glycerol kinase (glpK), and a protein of unknown function
(glpX) (31, 32). It was initially reported that GlpX displays
limited sequence similarity to the Synechococcus leopoliensis
fructose 1,6-bisphosphatase (FBPase) (31). In our work, a
more recent BLAST search revealed that GlpX manifests 39%
identity to an FBPase of Synechococcus sp. strain PCC7492
(29). Until now, the only recognized E. coli FBPase was en-
coded by fbp (25). This FBPase (FBPase I) has only 10%
identity to the amino acid sequence of glpX-encoded FBPase
(FBPase II). E. coli FBPase I is dependent on Mg2?, is inhib-
ited by low levels of AMP, is tetrameric (1), and is necessary
for growth of E. coli on gluconeogenic substrates such as glyc-
erol or succinate (10).
It is not clear why E. coli would maintain two distinct FB-
Pases. FBPases can modulate the concentration of fructose
1,6-bisphosphate [Fru(1,6)P2] and fructose 6-phosphate. These
two regulatory hexoses affect glycolysis enzymes 6-phospho-
fructokinases I and II, pyruvate kinase I, and phosphoenol-
pyruvate (PEP) carboxylase (2, 4, 13, 20); glycogen synthesis
enzyme ADP-glucose pyrophosphorylase (12); and carbon-
source import pathway enzymes glycerol kinase and 1-phos-
phofructokinase (6, 15). Flux through the Embden-Meyerhof
pathway in the direction of glycolysis or gluconeogenesis can
be allosterically controlled at the enzyme level by other me-
tabolites as well: PEP, ATP, ADP or AMP (9). The potential
“futile cycle” of phosphofructokinases and FBPases is also
alleviated by this regulation. Therefore, regulation of FBPases
In this communication, the FBPase activity of the glpX gene
product is documented. The glpX-encoded enzyme, FBPase II,
was purified and characterized, enabling comparison of the
attributes of E. coli FBPases in vitro. Further, a chromosomal
insertion mutation in glpX was constructed to test the physio-
logical effects of the glpX mutation on carbohydrate metabo-
E. coli strains and cloning of glpX. E. coli strains used in this
study are listed in Table 1. Strains were grown in Luria broth
(LB) supplemented with antibiotics as needed or in minimal
medium (7) containing 0.4% glycerol or 0.2% glucose.
The glpX gene was PCR amplified from chromosomal DNA
of strain MG1655 using the primer pair acgtgaaTTCCCCTG
TGCTACACTTCG (hybridizes to a region 48 bp upstream of
the initiation codon) and acgttctagaTTGCCTGTTACCCAAT
CAGC (hybridizes to a region 102 bp downstream of the ter-
mination codon), containing sequence mismatches (lowercase)
and restriction sites EcoRI and XbaI (underlined). The PCR
product was ligated into XbaI/EcoRI-cut pZE14 (18) forming
pJB100. This plasmid was introduced into strain DF657 (?fbp)
to test for complementation of the Fbp?(glycerol-negative)
phenotype. DF657(pJB100) was capable of slow growth on
glycerol. Therefore, increased expression of glpX from the
high-copy-number plasmid pJB100 complements the Fbp?
Overexpression and purification of FBPase II. To facilitate
overexpression and purification of FBPase II, a pT7-7 (28)
derivative containing glpX was constructed (pJB300B). The
NdeI-SalI fragment that was inserted into the same sites of
pT7-7 to form pJB300B was obtained by PCR amplification
using primers acgtaccaattgaggagatatacaTATGAGACGAGAA
CTTGCCATC and acgtgtcgacTGCCTTATCTTCGTTCTC
CG, with DNA from strain MG1655 as the template (sequence
mismatches are lowercase and restriction sites are underlined).
The expected nucleotide sequence of glpX in pJB300B was
JB108(pJB300B) with 200 ?M isopropyl-?-D-thiogalactopyr-
anoside (IPTG) for 2 h in a 200-ml LB culture with an optical
density at 600 nm of 0.6. Cells were harvested by centrifuga-
tion, resuspended in 2.5 ml of 20 mM Tricine (pH 7.7)–50 mM
KCl–1 mM MgCl2–1 mM dithiothreitol (DTT)–0.5 mM
EDTA, lysed by sonication, and centrifuged at 140,000 ? g for
30 min. Nucleic acids were precipitated by addition of poly-
ethylenimine (0.05%, vol/vol) and centrifugation at 10,000 ? g
for 20 min. FBPase II activity was precipitated by addition of
0.4 g of (NH4)2SO4ml?1. The (NH4)2SO4pellet was dissolved
in 2.3 ml of 50 mM Tris-HCl (pH 7.7)–0.2 mM MgCl2–0.1 mM
EDTA and fractionated by anion exchange chromatography
on a Q HR15 column (Waters) at room temperature using a
gradient of 0.075 to 0.5 M NaCl in 50 mM Tris-HCl (pH
7.7)–0.2 mM MgCl2–0.1 mM EDTA. Peak fractions were
pooled and precipitated with 0.5 g of (NH4)2SO4ml?1, redis-
* Corresponding author. Mailing address: Department of Biochem-
istry, Virginia Polytechnic Institute and State University, Blacksburg,
VA 24061. Phone: (540) 231-7060. Fax: (540) 231-9070. E-mail: tilarson
solved, and then dialyzed against 20 mM Tricine (pH 7.7)–1
mM MgCl2–0.1 mM DTT–15% glycerol. All steps of the pu-
rification procedure were monitored by coupled spectrophoto-
metric assay. A total of 2.8 mg of protein was purified 4.4-fold,
with a 55% recovery and specific activity of 4.2 U mg?1. Anal-
ysis of purified product by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis showed that the enzyme was ?95%
pure. Protein concentrations were determined as described by
Bradford (5) with bovine serum albumin as the standard.
Optimal conditions for enzyme activity. Using a coupled
spectrophotometric assay (Fig. 1), the highest activity was ob-
tained with 8 ?g of FBPase II in a 1-ml reaction mixture
containing 0.5 to 2 mM MnCl2, 50 to 125 mM KCl, 0.02 M
Tricine (pH 7.7), and 1.5 mM Fru(1,6)P2. Omission of Mn2?
resulted in ?90% loss of activity. Replacing Mn2?with 1 mM
ZnCl2, CaCl2, FeSO4, or CuSO4resulted in almost complete
loss of activity. Replacement with 3 to 10 mM MgCl2resulted
in ?10% of the activity present with Mn2?. The presence of 1
mM DTT had no effect on FBPase II activity.
Molecular mass determination. The molecular mass of the
GlpX subunit estimated by using sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis was 40 kDa, which is near the
expected size based on the deduced amino acid sequence (36
kDa). Gel filtration chromatography was performed using a
Waters Protein Pak Glass 300SW column in 20 mM Tricine
(pH 7.7)–100 mM KCl with 1 mM MnCl2or 1 mM MgCl2.
Standards included ribonuclease A (17 kDa), carbonic anhy-
drase (29 kDa), ovalbumin (45 and 90 kDa), bovine serum
albumin (66 and 132 kDa) and phosphorylase b (97.4 kDa).
Times of protein elution were determined by monitoring the
absorbance at 280 nm. At high, medium, and low concentra-
tions of FBPase II (320, 160, and 40 ?g of FBPase II, respec-
tively, in 200 ?l of loading volume), the protein eluted at 90,
80, and 47 kDa, respectively. FBPase II is most likely a dimer
at higher concentrations, in contrast to the tetrameric fbp-
encoded FBPase I (1).
Catalytic properties. A Kmof ?35 ?M and Vmaxof ?3.3 U
mg?1were determined for FBPase II using Fru(1,6)P2levels
below 0.25 mM (Fig. 1). Substrate inhibition was observed at
high Fru(1,6)P2concentrations. FBPase II has lower affinity
for Fru(1,6)P2compared to FBPase I (apparent Kmof 5 ?M
). Based on specific activities of the purified enzymes, the
deduced turnover number for FBPase II is about seven times
less than that of FBPase I (19).
Substrate specificity of FBPase II. Substrate specificity was
determined by measuring the rate of enzyme-catalyzed pro-
duction of inorganic phosphate from various potential sub-
strates (see footnote d to Table 2). Fru(1,6)P2was the best
substrate found, while fructose 1-phosphate and ribulose 1,5-
bisphosphate served less well as substrates. The apparent Km
and Vmaxof FBPase II with fructose 1-phosphate were 1 mM
and 1.4 U mg?1, respectively, giving a Vmax/Kmalmost 70 times
lower than that obtained using Fru(1,6)P2. Ribulose 1,5-
bisphosphate at 1 mM produced 15% of the phosphatase ac-
tivity obtained with 1.5 mM Fru(1,6)P2. Glucose 6-phosphate,
fructose 6-phosphate, mannose 6-phosphate, glucose 1,6-
6-phosphate, and glycerol-P were not significant substrates (all
present at 1 mM [data not shown]).
Effectors of FBPase II. Fructose 1-phosphate, inorganic
phosphate, ADP, and ATP inhibited FBPase II activity (Table
2). Fructose 1-phosphate and inorganic phosphate inhibited
activity competitively, with apparent Kis of 1 and 0.35 mM,
respectively. Fructose, fructose 6-phosphate, and glucose
6-phosphate were not inhibitors (data not shown). AMP had
no effect on enzyme activity, but ATP and especially ADP
inhibited activity at low Fru(1,6)P2(?0.1 mM). PEP almost
doubled enzyme activity (Table 2), while addition of dihydroxy-
acetone phosphate, glycerol, or glycerol-P (all at 1 mM) did
not affect activity (not shown). The response of FBPase II to
effector molecules PEP, ADP, and ATP associates it with many
enzymes of the Embden-Meyerhof pathway that are regulated
TABLE 1. E. coli strains used in this study
StrainRelevant genotype Description and/or reference
Wild-type isolate (F?rph-1 ??)
F?zjg-920::Tn10 rph-1 ??
Hfr pfkA2 fhuA22 ompF627 (T2
spoT1 rrnB2 mcrB1 creC510
F?edd-1 gnd-1 gnt pps-1 ?argH1 iclR15(Con)
HfrC ?fbp287 fhuA22 spoT1 pit-10 relA1 ??ompF627(T2
BL21(DE3) zjg920::Tn10 ?fbp287
TL524 glpX::Spcr?fbp287 zjg-920::Tn10
TL524 glpX??fbp287 zjg-920::Tn10
TcSderivative of TL504 (22)
r) fadL701 (T2
r) relA1 pit-10
P1(CAG12019) into DF657
P1(JB100) into BL21(DE3) (27)
P1(CAG12185) into JLD1001, select Tcr, score Spr
P1(JLD1101) into TL524
P1(JLD1301) into TL524, select Spr, score Tcr
P1(JB100) into JLD2401
P1(JB100) into JLD2401
P1(JB100) into TL524
P1(JB100) into TL524
aThe ?fbp287 deletion affects not only fbp but also the flanking genes yjfF, yjfG, ytfT, and ytfS.
bConstruction of the glpX insertion mutation: strain JLD1001 was formed by insertion of the Spcrgene into the BsiWI site of glpX, using a plasmid or chromosomal
integration selection system (16). pBS-glpX was constructed from SacI/BamHI-digested pBluescript KS(?) (Stratagene) ligated to the SacI/BamHI ?3,400-bp fragment
of pDW23 (32) encompassing the glpK, glpX, and fpr genes. Plasmid pBS-glpX::Spcrwas constructed by ligation of SmaI-excised Spcrgene, from pHP45? (8), into the
filled-in (now blunt) BsiWI site of glpX in pBS-glpX. pKO3-glpX::Spcrwas formed by ligation of the SacI (blunted)/SalI piece of pBS-glpX::Spcrwith SmaI/SalI-digested
pKO3 (16). Plasmid pKO3-glpX::Spcrcontains genes glpK, glpX::Spcr, and fpr. From pKO3-glpX::Spcr, glpX::Spcrwas integrated into strain TL524 to form strain
JLD1001. The integration site was verified by P1 transduction of JLD1001 to Tcr, with strain CAG12185 as the P1 donor, to form strain JLD1101. Comparison of PCR
and Southern analyses of DNA of strains JLD2402 and JLD2403, containing the insertional mutation, with JLD2404 and JLD2405 (glpX?), also confirmed integration
of the Spcrgene in the glpX gene (data not shown).
VOL. 182, 2000 NOTES5625
by PEP, Fru(1,6)P2, ATP, ADP, or AMP (9). The properties of
FBPase II are distinct from those of FBPase I, an enzyme
exquisitely sensitive to inhibition by AMP (50% inhibition by
15 ?M AMP ). AMP inhibition of FBPase I is alleviated by
PEP, although higher concentrations of PEP are inhibitory
(?1 mM PEP).
Physiological role of glpX. Genetic studies were undertaken
in an attempt to discern the physiological role of glpX. First,
the roles of the two FBPases were investigated using congenic
strains with the four possible combinations of wild-type and
defective fbp and glpX alleles: JLD2402 (?fbp glpX::Spcr),
JLD2403 (fbp?glpX::Spcr), JLD2404 (?fbp glpX?), and
JLD2405 (fbp?glpX?) (Table 1). The fbp?glpX::Spcrstrain
grew as well as the wild type on LB or glucose, fructose,
succinate, or glycerol minimal medium, aerobically or anaero-
bically. Apparently, FBPase II is not crucial to cell growth
under these conditions when FBPase I is present. Strains lack-
ing FBPase I (e.g., ?fbp glpX?strains DF657 and JLD2404)
grew normally on glucose or fructose medium but were unable
to grow on minimal medium supplemented with glycerol or
other gluconeogenic substrates, indicating that chromosomal
glpX?does not compensate for the loss of fbp expression.
(However, increased expression of glpX from multicopy plas-
mid pJB100 complemented the Fbp?phenotype.)
The ?fbp glpX?strain (JLD2404) was able to revert to slow
growth on glycerol minimal medium enriched with 0.03%
Casamino acids and 2.5 mM KNO3(with or without O2).
These revertants lost the ability to grow on glycerol when
transduced to glpX::Spcr. The ?fbp glpX::Spcrstrain JLD2402
did not show any reversion under the same conditions. A
possible explanation for reversion of ?fbp glpX?strains to a
glycerol-positive phenotype is a mutation yielding elevated
The effect of the glpX and fbp mutations on glycogen accu-
mulation was tested by exposure of the four congenic strains to
iodine vapor following growth on Kornberg medium contain-
ing 0.4% glycerol instead of glucose (17). Glycogen accumu-
lated to higher levels in both fbp?strains (maroon colony
color) compared to levels found in the ?fbp mutants (orange
colony color). Therefore, accumulation of glycogen to wild-
type levels requires FBPase I but is not affected by the glpX
We hypothesized that the inability of ?fbp glpX?strains to
grow on gluconeogenic substrates may be due to low-level
expression of glpX combined with competing hexose catabolic
pathways that would prevent accumulation of hexose sufficient
for cell growth. To test this possibility, the ?fbp allele was
introduced by cotransduction with zjg920::Tn10 into a pfkA2
strain deficient in glycolysis (phosphofructokinase I; strain
AM1) and into an edd gnd strain deficient in the Entner-
6-phosphogluconate dehydrogenase; strain R6). In both cases,
transductants unable to grow on glycerol were obtained at the
expected frequency, demonstrating that glpX function is insuf-
ficient to provide hexose for growth of these strains where
presumed competing pathways are impaired. We did observe
that a pfkA glpX::Spcrstrain grew more slowly with glucose or
glycerol than did the pfkA glpX?parent strain AM1, indicating
a functional importance of FBPase II in this strain.
FIG. 1. (A) Dependence of FBPase II activity on concentration of
Fru(1,6)P2. Each data point is presented as an average of two to four determi-
nations, with standard errors (error bars). Coupled spectrophotometric assay was
used with a solution containing 8 ?g of FBPase II, 0.2 U of phosphoglucose
isomerase, 0.4 units of glucose-6-phosphate dehydrogenase, 20 mM Tricine (pH
7.7), 50 mM KCl, 1 mM MnCl2, and 0.25 mM NADP in a final volume of 1 ml
at room temperature (24). The rate of reduction of NADP to NADPH was
determined by monitoring absorbance at 340 nm. (B) Lineweaver-Burk replot of
selected data points from panel A.
TABLE 2. Effectors of FBPase II with Fru(1,6)P2substrate
Effector (concn [mM])Reaction rate (%)
Mn-AMP (1)............................................................................. 100a
Mn-ADP (1.25)......................................................................... 50b
Mn-ADP (1.9)........................................................................... 38b
Mn-ATP (1.25) ......................................................................... 88b
Fructose 1-phosphate (2) ........................................................
PEP (1)...................................................................................... 170d
aEnzyme activity was determined by coupled spectrophotometric assay con-
taining 8 ?g of FBPase, 0.05 mM Fru(1,6)P2, and effector. Reaction rates were
compared to the rate with no effector added, which was 2.3 U mg?1for this
bEnzyme activity was determined by coupled spectrophotometric assay con-
taining 8 ?g of FBPase, 0.05 mM Fru(1,6)P2, effector, and Mn2?in 1 mM excess
of effector concentration. Reaction rates were compared to the rate with no
effector added, which was 1.5 U mg?1for this preparation.
cEnzyme activity was determined as explained in footnote b but with 1 mM
dEnzyme activity was determined by inorganic phosphate quantitation assay,
containing 2 ?g of FBPase, 0.6 mM Fru(1,6)P2, and PEP. Reaction rates were
compared to the rate with no effector added, which was 2.3 U mg?1. Reaction
mixtures (0.5 ml) containing 20 mM Tricine (pH 7.7), 50 mM KCl, 1 mM MnCl2,
Fru(1,6)P2, and enzyme were incubated at room temperature. Aliquots were
removed for phosphate determination using the malachite green-ammonium
molybdate reagent (14). One unit of enzyme activity is defined as the amount of
enzyme catalyzing the formation of 1 ?mol of product in 1 min.
Three classes of FBPases. Bacterial FBPases are members
of three different orthologous groups (30), including a group of
glpX-encoded FBPases (class II). Some bacteria that contain
orthologs of FBPase II include Klebsiella aerogenes (91% iden-
tity), Yersinia pestis (81%), Haemophilus influenzae (67%), Ba-
cillus subtilis (49%), Clostridium acetobutylicum (45%), Myco-
bacterium tuberculosis (43%) and Synechococcus sp. strain
PCC7942 (39% ). There is little similarity of these class II
FBPases to the other two orthologous groups of FBPases,
those of FBPase I (E. coli) (class I), and those of a very
divergent FBPase of B. subtilis (class III ). From the se-
quence data available, no bacterial genome has a combination
of class I and class III FBPases, although combinations of
classes I and II and of classes II and III are seen.
In conclusion, glpX-encoded FBPase II is still somewhat of
an unknown entity in the overall framework of E. coli metab-
olism. FBPase II is conserved in many other bacteria and is the
only known FBPase in some organisms (e.g., M. tuberculosis).
The enzyme is distinct in many aspects from FBPase I. How-
ever, the specificity and high affinity of FBPase II for substrate
and its regulation by PEP and ADP suggest that the enzyme
functions with FBPase I in the central pathways of carbohy-
We thank G. Church for providing the pKO3 plasmid system and Ali
T. van Loo-Bhattacharya for technical support.
This work was supported in part by NSF grant MCB-9118757.
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