(Received for publication, May 3, 1995)
From the
A multidomain protein comprising the four subunits of the
glucose phosphotransferase system of Escherichia coli was
constructed by fusion of the transmembrane subunit IICB
The enzymes of a metabolic pathway can interact with each other
to form highly organized enzyme complexes (Reed, 1974; Perham, 1975;
Srivastava and Bernhard, 1986a, 1986b; Srere, 1987; Perham, 1991).
These interactions range from weak and transient to covalent as found
in multifunctional enzymes (Srere, 1985, 1987). For the enzymes in the
tricarboxylic acid cycle, interactions have been demonstrated between
six and eight sequential enzymes, and all of the enzymes have been
found to bind to the inner surface of the mitochondrial inner membrane
(D'Souza and Srere, 1983; Robinson and Srere, 1985; Datta et
al., 1985; Brent and Srere, 1987). Five of the tricarboxylic acid
cycle enzymes of Escherichia coli can also be isolated as a
high molecular weight complex (Barnes and Weitzman, 1986). There are
several possible reasons for such organization, including enhancement
of catalytic activity and substrate channeling. A complex of
functionally related enzymes might be more efficient in catalyzing a
multistep metabolic sequence than would be a collection of independent
enzymes. In other words, complex formation provides a means of
concentrating catalysts rather than having them randomly distributed in
the cell. Complex formation also offers a way of segregation of enzymes
that would otherwise compete for the same intermediate. Such metabolic
channeling has been invoked frequently in speculations on the role of
multienzyme complexes in cellular economy, and has been suggested to be
important in many cases, for instance, in shortening the transient
times of overall reactions in metabolic processes (Welch and Easterby,
1994). The bacterial phosphotransferase system (PTS) ( The sequential phosphoryltransfer between
the different proteins necessitates protein-protein interactions. It
has been suggested that the components of the PTS exist as
membrane-associated multiprotein complexes rather than as individual
components (Scholte et al., 1982; Brouwer et al.,
1982; Saier et al., 1982; Misset et al., 1983). The
actual composition of the complexes might vary, depending on the
metabolic requirements of the cell. Steady-state kinetic studies with
PTS proteins of E. coli have revealed a K Fusion of sequential
enzymes by molecular biological techniques has been proposed as a means
for the study of proximity effects of complexes that tend to dissociate
due to dilution during isolation (Blow and
Mosbach, 1991). A number of bifunctional fusion proteins that catalyze
sequential reactions have been constructed (Blow,
1987; Ljungcrantz et al., 1989; Lindbladh et al.,
1994). The kinetic benefits of such fusions were shorter transient
times, and an apparent sequestering of the intermediates by the fusion
protein when compared with the system of free enzymes. Examples of
successful fusions are the fusions of yeast mitochondrial citrate
synthase and malate dehydrogenase (Lindbladh et al., 1994),
the fusion of subunits I, II and III of the cytochrome b0 ubiquinol oxidase of E. coli (Ma et al., 1993),
and the fusions between cytochrome P450 and the flavoprotein domain of
the NADPH P450 reductase of homologous as well as heterologous origin
(Fisher et al., 1992; Shet et al., 1994). Here we
characterize a fusion protein containing the four protein subunits
IICB
Figure 1:
Plasmid map
of pJFGCHI, linker sequence, and polyacrylamide gel of fusion protein. A, plasmid pJFGCHI encodes the fusion protein
IICB
Figure 2:
Phosphotransferase activity of the
purified fusion protein and of an equimolar mixture of the isolated
subunits. A, P-enolpyruvate-dependent phosphorylation of
Figure 3:
Dependence of phosphotransferase activity
on Triton X-100/lipid ratio. The concentration of the fusion protein
was 20 nM. Shown are means and S.D. of three independent
experiments. Assay condition were as follows:
[
Figure 4:
Effects of extra IICB
Fusion proteins
consisting of sequential enzymes can direct the substrate flow along
the linked active sites and protect it from diversion into other
metabolic pathways. In the phosphotransferase system, the
phosphorylcarrier protein HPr acts as a branch point of
phosphoryltransfer to the IIA domains of other PTS carbohydrate
transporters. Is the HPr moiety in the GCHI fusion protein still
accessible to free IIA units other than the covalently linked
IIA
Figure 5:
Channeling of phosphoryl transfer in the
fusion protein. Phosphorylation of [
Because the IICB Fusion of the four subunits resulted in
channeling of phosphoryltransfer such that the flow of phosphorylgroups
could no longer be diverted from the HPr domain of the GCHI fusion
protein toward the
IIAB Unexpectedly, the
phosphotransferase activity of the GCHI fusion was higher in a mixture
of Triton X-100 and phospholipids than in either of the two components.
Phospholipids are necessary for maximal activity of the transmembrane
IICB
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and the three cytoplasmic proteins, IIA
, HPr, and
enzyme I. The subunits were linked in the above order with Ala-Pro-rich
linkers; the fusion protein was overexpressed in E. coli and
purified by Ni
chelate affinity chromatography.
Approximately 3 mg of the fusion protein could be purified from 1 liter
of culture. The phosphotransferase activity of the purified fusion
protein was 3-4 times higher than that of an equimolar mixture of
the isolated subunits. The mannose transporter, which also requires
enzyme I and HPr, was not an effective competitor in the overall
phosphoryltransfer reaction when the fusion protein was used, whereas
it was a competitor when an equimolar mixture of the separate subunits
was employed. Transphosphorylation activity of the fusion protein was
almost indistinguishable from the wild-type IICB
.
Addition of extra IICB
subunit could significantly
stimulate the phosphotransferase activity of the fusion protein,
addition of extra IIA
subunit and enzyme I, in contrast,
was slightly inhibitory, and HPr had almost no effect. An optimal
detergent-lipid ratio is required for maximum activity of the fusion
protein. Our results suggest that Ala-Pro-rich linker sequences may be
of general use for the construction of catalytically active fusion
proteins with novel properties.
)comprises multienzyme complexes that are composed of
membrane-embedded subunits and cytoplasmic proteins. The system as a
whole has at least three functions, uptake of carbohydrates coupled to
their phosphorylation, regulation of metabolic pathways (catabolite
repression, inducer exclusion), and chemotactic signaling (for reviews
see Meadow et al.(1990) and Postma et al.(1993)). The
glucose-specific phosphotransferase system of E. coli in
particular consists of the membrane-embedded IICB
subunit
and of the three soluble subunits, IIA
, HPr, and enzyme
I. The four subunits form a phosphorylation cascade, which in the order
P-enolpyruvate
enzyme I
HPr
IIA
IICB
Glc transfers phosphorylgroups
from P-enolpyruvate to the transported sugar (Glc). Enzyme I is a
homodimeric protein (subunit M
63,489) of as yet
unknown structure. The enzyme I monomer consists of two equally sized
domains (Neyroz et al., 1987; Han et al., 1990;
LiCalsi et al., 1991). The carboxyl-terminal domain catalyzes
phosphoryltransfer from phosphoenolpyruvate to His
located on the amino-terminal domain (Alpert et al.,
1985; Saffen et al., 1987). HPr is a two-layer
/
protein (M
9109), which is phosphorylated on
His
in the loop between
1 and
1 (Jia et
al., 1994). Amino and carboxyl termini are at the same edge but on
opposing ends of the
-sheet with the carboxyl terminus close in
space to the active site histidine. IIA
(M
18,556) is a
-sheet sandwich with six antiparallel strands
on either side (Worthylake et al., 1991). The phosphorylatable
His
is located in the center of a shallow depression. The
amino and carboxyl termini of this monomeric subunit are close in
space. They are near the amino and carboxyl ends of the two terminal
-strands, which are direct neighbors in an antiparallel
-sheet. Besides serving as a phosphorylcarrier between HPr and
IICB
, IIA
has regulatory functions.
Unphosphorylated IIA
binds and inhibits other
transporters such as the lactose carrier and enzymes participating in
the carbohydrate metabolism such as glycerol kinase (Postma et
al., 1993; Hurley et al., 1993; Saier and Reizer, 1994).
Phosphorylated IIA
activates adenylcyclase by an unknown
mechanism. IICB
is a homodimeric protein (subunit M
50,647), which catalyzes vectorial translocation
and phosphorylation of glucose. The IICB
monomer consists
of two domains. The amino-terminal IIC domain (residues 1-386) is
predicted to span the membrane 8 times (Buhr and Erni, 1993). It
contains the sugar binding site. The carboxyl-terminal IIB domain
(residues 391-477) is a two layer
/
protein (Golic
Grdadolnik et al., 1994). (
)It becomes
phosphorylated on Cys
in the loop between
1 and
2 (Meins et al., 1993). The IIC and IIB domains can be
expressed as independent protein subunits that in combination retain
catalytic activity (Buhr et al., 1994) or can be circularly
permuted, affording a ``IIBC
'' protein with
catalytic activity comparable to wild-type IICB
. (
)This indicates that the amino and carboxyl termini of
IICB
both are on the cytoplasmic face of the membrane and
possibly close in space.
of 5 µM for the interaction between enzyme I
and HPr and of 0.3 µM for the interaction of phospho-HPr
with IIA
. In contrast, the K
of IICB
for IIA
exceeds 20
µM (Reizer et al., 1992; Postma et al.,
1993). Autophosphorylation of enzyme I with phosphoenolpyruvate is
enhanced strongly in the presence of HPr, indicating a stabilizing and
cooperative interaction between phospho-HPr and phosphoenzyme I (Erni,
1986). Similarly, IIA
increases the rate of
phosphoryltransfer from phospho-IICB
to glucose and slows
down spontaneous hydrolysis of phospho-IICB
(Erni, 1986).
However, the evidence for membrane-associated complexes is
circumstantial and they could not be isolated.
, IIA
, HPr, and enzyme I of the
glucose-specific phosphotransferase system. To facilitate the domain
movement required for active site coupling and optimal catalytic
activity, a naturally occurring linker peptide was used to connect the
subunits. Numerous oligopeptide sequences linking domains in protein
tertiary structures have been described (Erni, 1989; Argos, 1990;
Perham, 1991). From these, the Ala-Pro linker was chosen that occurs in
the decarboxylases and dehydrogenases, where it links the biotinylated
and lipoylated domains, respectively (Perham, 1991) and in proteins of
the bacterial phophotransferase system (Erni et al., 1987;
Peri and Waygood, 1988; Wu et al., 1990).
Bacterial Strains, Plasmids, and Growth
Media
Strain ZSC112LHIC is glk ptsG manZ ptsH ptsI
crr. It was constructed by conjugative transfer into ZSC112L
(Curtis and Epstein, 1975) of the ptsH ptsI crr deletion from
strain UE7 (Hfr KL16 thi
(ptsH ptsI crr) galR galP::Tn10;
a gift of W. Boos, University of Konstanz, Konstanz, Germany). The
expression vector pJF119EH contains lacI
and Ptac (Frste et
al., 1986), pTSG11 encodes IICB
(Buhr et
al., 1994) under the control of the Ptac promoter, and
pTSHIC9 encodes HPr, enzyme I, and IIA
also under the
control of the Ptac promoter (Mao et al., 1995).
Cells were grown at 37 °C in LB medium containing 0.1 mg/ml
ampicillin.
Plasmid Construction
The plasmid pJFGCHI was
assembled stepwise using the expression vector pJF119EH and coding
regions taken from plasmids pTSG11 and pTSHIC9. One DNA duplex encoding
the Ala-Pro-rich linker I (see Fig. 1B) was taken from
plasmid pTSL23 (Erni et al., 1987). The other duplex encoding
linker II (Fig. 1B) from the Rhodobacter capsulatus multiphosphoryl transfer protein (Wu et al., 1990) was
chemically synthesized. Gapped duplex mutagenesis (Stanssens et
al., 1989) and polymerase chain reaction site-directed mutagenesis
(Mikaelian and Sergeant, 1992) were used to introduce compatible
restriction sites at the ends of the sequences to be assembled.
Standard procedures were used for ligation, transformation, restriction
analysis, and plasmid preparation (Sambrook et al., 1989).
Double-stranded plasmid DNA was sequenced by the dideoxynucleotide
chain termination method using the Sequenase® kit (U. S.
Biochemical Corp.). Intermediates in the stepwise assembly of pJFGCHI
were the plasmids pJFGC encoding a fusion protein between IICB and IIA
with linker I, pJFCH encoding a fusion
protein between IIA
and HPr with linker II, and pJFHI
encoding a fusion protein between HPr and enzyme I with linker I
(Schunk, 1992; Gerber, 1993). (
)To facilitate purification,
a hexahistidine tag was introduced at the carboxyl-terminal end of the
fusion protein by polymerase chain reaction.
-IIA
-HPr-enzyme I under control of the Ptac promoter. Structure genes are indicated with solid
boxes, and segments encoding the three Ala-Pro-rich linkers are
indicated with open boxes. B, amino acid sequences of
the three linker regions are indicated in boldface. Of the
four subunits, only the amino-terminal and carboxyl-terminal sequences
are shown. Residues in lower case indicate His
Phe
substitutions. Residues in square brackets are present in the
wild-type protein but deleted in the fusion. C, lane1, molecular weight markers; lane2,
membranes (100 µg); lane3, proteins removed by
washing with Brij 35 (80 µg); lane4, Triton
X-100-insoluble fraction (10 µg); lane5, Triton
X-100-soluble fraction (5 µg); lane6, the fusion
protein (2 µg) after Ni
-nitrilotriacetic acid
purification.
Purification of the
IICB
E. coli ZSC112L-IIA
-HPr-Enzyme I Fusion
Protein
HIC(pJFGCHI) was grown
in 1 liter of LB at 37 °C to A
= 1.5
and induced with 50 µM
isopropyl-1-thio-
-D-galactopyranoside, and incubation
continued overnight. Cells were harvested by centrifugation (16,000
g at 4 °C for 10 min). The cell pellet (6 g) was
resuspended in 18 ml of buffer A (10 mM Tris/HCl, pH 7.5, 500
mM NaCl, 1 mM
-mercaptoethanol, 1 mM PMSF). Cells were broken by two passages through a French pressure
cell. Cell debris was removed by low speed centrifugation (12,000
g at 4 °C for 10 min). Membranes were collected by
high speed centrifugation (200,000
g at 4 °C for 2
h) and resuspended in buffer B (10 mM Tris/glycine, pH 9.3, 1
mM
-mercaptoethanol, 1 mM PMSF; 1 ml/g of
original cell paste). Brij 35 (Fluka, Buchs, Switzerland) was added to
a final concentration of 2% to the stirred suspension, and, after 10
min of additional stirring, the membranes were collected by high speed
centrifugation. The supernatant was discarded, and the membrane
sediment was resuspended in buffer B (1 ml/g of original cell paste).
These Brij 35-washed membranes were solubilized in 2% Triton X-100
(Serva, Heidelberg, Germany). After high speed centrifugation, the
membrane sediment was extracted a second time under the same
conditions. The pooled supernatants containing the solubilized fusion
protein were mixed with 4 ml of Ni
-nitrilotriacetic
acid resin (equilibrated with buffer C; 10 mM Tris/HCl, pH
8.2, 500 mM NaCl, 1 mM
-mercaptoethanol, 1
mM PMSF, 0.1% Triton X-100) and shaken for 1 h at 4 °C.
The resin with the adsorbed protein was packed into a column, washed
with 30 ml of buffer C and 30 ml buffer D (10 mM Tris/HCl, pH
7.5, 500 mM NaCl, 1 mM
-mercaptoethanol, 1
mM PMSF, 0.1% Triton X-100, 20 mM imidazole) until A
returned to base line. The fusion protein was
then eluted with buffer E (10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM
-mercaptoethanol, 1 mM PMSF,
0.1% Triton X-100, 100 mM imidazole). The flow rate was set to
0.5 ml/min, and 2-ml fractions were collected. The fractions containing
the fusion protein were dialyzed against buffer F (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1
mM dithiothreitol, 1 mM PMSF, 0.1% Triton X-100)
overnight at 4 °C.
Purification of Wild-type PTS Proteins
Enzyme I,
HPr, and IIA were purified as described by Mao et al. (1995), IICB
was purified as described by Waeber et al.(1993), and IIAB
was purified as described
by Stolz et al.(1993). IIC
-IID
was
purified by metal chelate affinity chromatography. (
)
Assay for Phosphoenolpyruvate:Sugar
Phosphotransferase Activity
Phosphotransferase activity was
assayed by the ion-exchange method of Kundig and Roseman(1971) as
described by Erni et al.(1982). The concentrations of PTS
proteins, of E. coliL--phosphatidylethanolamine
(Type IX, Sigma), and of Triton X-100 are indicated in the figure
legends.
Other Techniques
Protein samples were not boiled
in sample buffer prior to electrophoresis. The
IICB-IIA
-HPr-enzyme I fusion protein was
analyzed on a standard 10% polyacrylamide gel and stained with
Coomassie Blue (Erni et al., 1982). For immunoblots (Towbin et al., 1979), monoclonal anti-IICB
antibodies
(Meins et al., 1988) and alkaline phosphatase coupled to
anti-mouse IgG were used. Protein concentrations were determined by a
modified Lowry assay (Markwell et al., 1978) with bovine serum
albumin as standard.
Construction, Overexpression, and Purification of the
IICB
The four genes ptsG crr
ptsH, and ptsI were linked in the order by which their
respective gene products mediate phosphoryltransfer. To maximize
protein expression and minimize plasmid loss during fermentation, the
gene was put under the control of the Ptac promoter (Fig. 1A). The four protein subunits were linked
through Ala-Pro-rich linker peptides of 20 residues (Fig. 1B) taken from the IIAB-IIA
-HPr-Enzyme I Fusion
Protein (GCHI Fusion)
subunit of
the E. coli mannose transporter (Erni et al., 1987)
and from the multiphosphoryl transfer protein subunit of the R.
capsulatus fructose transporter (Wu et al., 1990). The
GCHI fusion could be solubilized from Brij 35-washed membranes. As
observed during purification of the mannose transporter, Brij 35
removed a large amount of other membrane (associated) proteins (Rhiel et al., 1994). 60% of the activity could be solubilized by
extracting the membrane fraction twice with 2% Triton X-100. The fusion
protein was purified to almost homogeneity by metal chelate affinity
chromatography on a Ni
-nitrilotriacetic acid column (Fig. 1C). Recoveries of 40% could be achieved using
batch absorption instead of loading the extract to a prepacked column.
3 mg of pure GCHI protein were obtained from 6 g, wet weight, of cells.
The fusion protein has a polyacrylamide gel electrophoretic mobility
corresponding to a molecular mass of 140 kDa, which is in good
agreement with the expected 150 kDa (Fig. 1C). The
stability (enzymatic activity) of the fusion protein could be improved
when imidazole used for elution was removed by dialysis. The
column-bound fusion protein could be washed with and then eluted in
detergent-free buffers without precipitation. The phosphotransferase
activity in detergent-free buffer was only 2%. 70% of the full activity
could, however, be recovered when detergent and lipids were added back
to the incubation mixture. Attempts to concentrate the fusion protein
in detergent-free buffer resulted in precipitation, indicating a
limited solubility under these conditions.
Phosphotransferase Activity of the GCHI
Fusion
E. coli ZSC112LHIC(pJFGCHI) fermented
glucose on MacConkey plates, indicating functional complementation of
the glucose phosphotransferase system by the fusion protein. The
phosphoenolpyruvate-dependent glucose phosphorylation activity of the
purified fusion protein is 3-4 times higher than that of an
equimolar mixture of the isolated subunits (Fig. 2A).
The nonlinear increase of activity with protein concentration is caused
by Triton X-100, which is introduced with each aliquot of fusion
protein (Fig. 2B). It does not reflect an exponential
protein-activity relationship. The GCHI fusion protein and the free
subunit system both require phospholipids for maximal activity, but the
fusion protein is further stimulated by low concentrations of
detergent. Fig. 3shows that a detergent lipid mixture is better
than either pure phospholipid or pure detergent, while the free subunit
system is inhibited by increasing concentrations of detergent (Fig. 2B). An optimal detergent-lipid ratio is required
for maximum activity of the fusion protein. The GCHI fusion also
catalyzes equilibrium exchange of phosphate between glucose and glucose
6-phosphate with 80% of the efficiency of the IICB
subunit (Fig. 2C), indicating that
IIA
, HPr, and enzyme I in the fusion protein do not
strongly interfere with substrate binding. In this case, however, the
nonlinear protein-activity relationship is due to the inhibitory effect
on transphosphorylation of increasing Triton X-100 concentrations
(results not shown).
MG by the fusion protein (
) and by the subunit mixture
(
). Triton X-100 concentration increased from 0 to 0.009% for the
fusion protein, and from 0 to 0.003% for the subunit mixture. B, effects of Triton X-100 on P-enolpyruvate-dependent
phosphotransferase activity of the fusion protein (40 nM,
) and of the subunit mixture (40 nM of each subunit,
). 100% activity corresponds to 3.90 nmol of
MG-P/30 min for
the fusion protein and to 1.48 nmol of
MG-P/30 min for the subunit
mixture. C, glucose 6-phosphate-dependent transphosphorylation
of [
C]Glc by the fusion protein (
) and by
the purified IICB
subunit (
). Concentration of
Triton X-100 increased from 0 to 0.027% for the fusion protein and from
0 to 0.009% for the IICB
subunit. Shown are means and
S.D. of three independent experiments. Assay conditions were as
follows: E. coli lipids (Type IX, Sigma) 2 mg/ml;
[
C]
MG (DuPont NEN) 0.5 mM, 1000
dpm/nmol (A and B); [
C]Glc
(DuPont NEN) 1 mM, 550 dpm/nmol (C); glucose
6-phosphate 65 mM.
C]
MG 0.5 mM, 1000
dpm/nmol.
Channeling of Phosphoryl Transfer
The GCHI fusion
protein has a 4-fold higher catalytic activity than a 1:1:1:1 mixture
of the four subunits. This modest increase could be a consequence of
two opposing effects: (i) high local concentration of active sites
which accelerates phosphoryltransfer and (ii) steric constraints which
cause non-optimal docking between phosphorylation sites and decrease of
the phosphoryltransfer rate. To identify possible
``bottlenecks'' in the phosphoryltransfer chain,
``bypasses'' were opened by adding the subunits, one at the
time, to the fusion protein and, for comparison, also to the 1:1:1:1
subunit mixture. Addition of IICB had a 2-3-fold
stimulatory effect in both systems, suggesting that phosphoryltransfer
between IIA
and IICB
and/or between
IICB
and glucose is rate-limiting (Fig. 4). This
stimulatory effect is due to IICB
itself. It cannot be
accounted for by the increasing Triton X-100 concentration, which in
this range would stimulate no more than a few percent (Fig. 2B). In contrast, addition of IIA
and enzyme I in up to 7-fold molar excess to the fusion protein
is slightly inhibitory (Fig. 4A). The reason for this
effect is not understood. No inhibition occurs in the control assay
with the single subunits (Fig. 4B).
,
IIA
, HPr, and enzyme I on the phosphotransferase activity
of the fusion protein (A) and of the subunit mixture (B). The concentrations of IICB
(
),
IIA
(
), HPr (
), and enzyme I (
) are
indicated. A, 100% activity corresponds to 3.43 ± 0.54
nmol of
MG-P/30 min at 40 nM fusion protein. The Triton
X-100 concentration increased from 0.010 to 0.012% when IICB
was added. B, 100% activity corresponds to 1.03 ±
0.09 nmol of
MG-P/30 min at 40 nM subunit concentration.
Triton X-100 concentration increased from 0.003 to 0.005%. Shown are
means and S.D. of three independent experiments. Assay condition as in Fig. 2A.
moiety? The
IIAB
IIC
IID
complex and dGlc were added to the GCHI fusion and to the system
of free subunits. As shown in Fig. 5, the mannose transporter
complex did not interfere with the phosphotransferase activity of the
GCHI fusion but competitively inhibited the activity of the free
subunits. The 1.5-fold increase of the GCHI activity is due to the
Triton X-100, which is introduced together with IICD and not to IICD
itself. This suggests that channeling of the phosphoryl group transfer
by the GCHI fusion occurs and that phosphorylgroups in the fusion are
effectively protected from diversion into other phosphotransferase
cascades.
C]
MG by
the fusion protein (
) and by an equimolar mixture of the isolated
subunits (
) were shown. Activities of the fusion protein (40
nM) or the subunit mixture (40 nM of each subunit)
were compared in the presence of an increasing concentration of the
mannose transporter (IIAB
,
IIC
IID
) and 2.5 mM unlabeled
2-deoxyglucose. The concentration of Triton X-100 increased from 0.01
to 0.03% for the fusion protein and from 0.003 to 0.023% for the
subunit mixture. 100% activity corresponds to 4.07 ± 1.10 nmol
of
MG-P/30 min for the fusion protein and to 1.02 ± 0.04
nmol of
MG-P/30 min for the subunit mixture. Shown are means and
S.D. of three independent experiments. Assay condition as in Fig. 2A.
Length and Stability of Linker Peptides
The four
subunits of the glucose-specific phosphotransferase system were linked
in the order by which they catalyze the phosphoryltransfer from
phosphoenolpyruvate to glucose. The membrane-spanning IICB subunit was placed at the amino terminus in order to minimize
possible interference with membrane insertion. The subunits were linked
by Ala-Pro-rich linker peptides of 20 residues. If fully extended, this
linker can span a distance of 6.5 nm, corresponding to the
cross-section of spherical protein of M
110,000.
Ala-Pro linkers can function as universal joints between domains. They
adopt a relatively stiffened conformation and cannot collapse to a
random coil (Radford et al., 1989; Perham, 1991). While their
amino acid sequence is unlikely to be important, length and composition
may influence (i) proteolytic stability of the linker and (ii) activity
of the linked subunits. Only a few very faint protein bands of lower
electrophoretic mobility could be detected with a monoclonal
anti-IICB
antibody on Western blots of cell lysates
(results not shown). With a polyclonal anti-IIA
serum, a
stronger smear of cross-reacting degradation products could be detected
(results not shown) which, however, were removed by Ni
chelate affinity chromatography. Doubling of the linker length
(from 21 to 40 residues) between the IIA
and HPr domain
in the GCHI fusion resulted in markedly enhanced degradation by host
proteases (results not shown). However, susceptibility to proteolysis
is context-specific. A similar increase of the length of the linker was
well tolerated in the two-domain IIAB
subunit and the
binary IICB
-IIA
fusion protein (Erni et
al., 1989; Schunk, 1992). Taken together, the Ala-Pro-rich linker
appears rather stable against endogenous proteases, while it turned out
to be highly sensitive toward in vitro proteolytic cleavage
(Erni et al., 1989). The length of the linker also affects
phosphotransferase activity. Doubling its length increased the activity
of the binary HPr-enzyme I fusion 1.4-fold; removal decreased activity
0.5-fold. Doubling the length did not affect the activity of the binary
IICB
-IIA
fusion; removal decreased activity
to 17% of the control with a 20-residue linker (Schunk, 1992; Schunk et al., 1992).
Activity of Fusion Protein and Possible
Mechanism
The quaternary fusion protein is active in vivo and in vitro. The transport activity in vivo indicates that incorporation of the transmembrane IIC domain into
the fusion protein did not compromise membrane insertion. The purified
GCHI fusion has a 4-fold higher overall k than
an equimolar mixture of the four subunits. This rules out the idea that
the observed activity is only residual and due to proteolytic fragments
generated from the fusion protein during purification. The quaternary
fusion is more active than any of the binary fusions, suggesting a high
degree of cooperativity between the domains and a strong compensation
by proximity effects of potentially unfavorable steric constraints. For
comparison, the binary HPr-enzyme I fusion has only 10% of the activity
of an equimolar mixture of the separate subunits, the binary
HPr-IIA
fusion has less than 5%, and only the binary
IICB
-IIA
fusion has a 4-fold higher
activity than the 1:1 mixture (Schunk et al. 1992). The
phosphotransferase activities of the HPr-enzyme I and
IIA
-HPr fusions could be stimulated by in vitro tryptic cleavage, suggesting unfavorable steric constraints in the
fusion proteins (results not shown).
subunit occurs as a dimer (Erni et al., 1982; Meins et al., 1988) the fusion protein could dimerize, too. In this
case, phosphoryltransfer could occur either along the domains within a
monomer or between domains on different monomers. It is not known which
mechanism is operative. Indicative of an intersubunit mechanism would
be the negative dominance of a GCHI fusion with four inactive
phosphorylation sites.
IIC
IID
complex. The addition of enzyme I, HPr, or IIA
did
not stimulate the overall phosphotransferase activity of the GCHI
fusion protein, indicating that phosphoryl groups could not be funneled
into the fusion protein, either. In contrast, the binary HPr-enzyme I
fusion could be stimulated with extra HPr (Schunk et al.,
1992), and the binary IICB
-IIA
fusion could
be stimulated with extra IIA
(Schunk, 1992). However, no
stimulation of the binary IIA
-HPr fusion by extra HPr or
IIA
occurred. (
)
subunit (Erni et al., 1982), while the
detergent might prevent intramolecular aggregation of the crowded
domains by loosening hydrophobic contacts. That the fusion protein
tends to aggregate became manifest during attempts at keeping the GCHI
fusion in detergent-free buffers. The protein could be eluted from the
metal chelate column without detergent, but yields were less than 40%
and subjected to high speed centrifugation (200,000
g at 4 °C for 20 min) it immediately sedimented. The protein
also aggregated upon standing at 4 °C. Nevertheless, should the
hydrophilic part (IIB-IIA-HPr-enzyme I), which accounts for 70% of the
total mass, determine the behavior of the protein, working without
detergents might be an option in attempts to crystallize the fusion
protein.
Other Fusion Proteins of the Phosphotransferase
System
Although no phosphotransferase system comprising all four
phosphorylation sites and the transmembrane domain in a single
polypeptide has been found in nature, fusion proteins comprising two or
three modules exist. The most frequent natural fusions occur between
IIA and IICB, e.g. in the mannitol transporter
(IICBA) and the GlcNAc transporter
(IICBA
) (for reviews, see Erni(1992), Postma et
al.(1993), and Lengeler et al.(1994)). In
IICBA
, channeling between IIA and IIB is not tight,
and phosphorylgroups can also be transferred from the IIA domain of
IICBA
to the IIB domain of the glucose transporter
(IICB
) (Vogler et al., 1988). FPr of Salmonella typhimurium is a fusion protein comprising an HPr,
a IIA, and a third domain exhibiting 60% similarity to the consensus
receiver module of two-component regulatory systems (Geerse et
al., 1989; Wu et al., 1990). Being part of the fructose
transporter (IIBC
) FPr nevertheless can complement HPr
defects by transferring phosphorylgroups to IIA domains other than the
one of FPr (Geerse et al., 1989). The most spectacular of
naturally occurring fusion proteins is multiphosphoryl transfer protein
of R. capsulatus, which comprises the enzyme I, HPr, and IIA
domains belonging to the fructose transporter (Wu et al.,
1990).
Conclusion
A number of recombinant bifunctional
fusion proteins, which catalyze sequential reactions, have been
reported. Compared with the separate enzymes in solution, they showed
kinetic benefits for the overall reaction, e.g. shorter
transient times and sequestering of the intermediate
(Blow, 1987; Ljungcrantz et al., 1989;
Lindbladh et al., 1994). The fusion protein reported in this
paper, which consists of four independent but functionally related
proteins, is apparently more complicated than any other that has been
reported so far. It is not yet clear how the four components of the PTS
interact with each other. Studies on interactions between the
IIB and the IIA
subunits by NMR are now in
progress (Golic Grdadolnik et al., 1994). (
)Our
results are consistent with other observations on fusion proteins,
suggesting that gene fusion technique may be a general and powerful
method to construct fusion proteins that have novel properties.
Crystallization of the fusion protein could be facilitated if
IIA
and HPr, known to crystallize, tend to dominate the
behavior of the system and should the fusion protein behave more like a
soluble than like a membrane protein. From a practical point of view,
the use of artificial multifunctional enzymes (fusion proteins) will be
useful in metabolic engineering. Multifunctional enzymes will help to
direct substrates into a desired metabolic pathway in vivo.
The GCHI fusion protein containing all components necessary for
transport and phosphorylation is now being expressed in yeast to test
whether it will function in an heterologous environment where no
phosphotransferase system has been found.
, transmembrane subunit of
the glucose transporter; IIA
, cytoplasmic subunit of the
glucose transporter; HPr, histidine-containing phosphocarrier protein
of the PTS; PMSF, phenylmethanesulfonyl fluoride; GCHI fusion,
IICB
-IIA
-HPr-enzyme I
fusion protein; IIC
and IID
, transmembrane
subunits of the mannose transporter; IIAB
, hydrophilic
subunit of the mannose transporter; IICBA
, the N-acetylglucosamine transporter; ptsG ptsH ptsI crr,
genes coding for IICB
, HPr, enzyme I, and
IIA
;
MG, methyl-
-D-glucopyranoside.
We thank Dr. B. Stolz for advice and helpful
discussions during this work.
und HPr des Phosphotransferasesystems von Escherichia coli. University of Berne, Switzerland
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.