(Received for publication, January 2, 1996; and in revised form, February 21, 1996)
From the
malZ is a member of the mal regulon. It is
controlled by MalT, the transcriptional activator of the maltose
system. MalZ has been purified and identified as an enzyme hydrolyzing
maltotriose and longer maltodextrins to glucose and maltose. MalZ is
dispensable for growth on maltose or maltodextrins. Mutants lacking
amylomaltase (encoded by malQ), the major maltose utilizing
enzyme, cannot grow on maltose, maltotriose, or maltotetraose, despite
the fact that they contain an effective transport system and MalZ. From
such a malQ mutant a pseudorevertant was isolated that was
able to grow on maltose. The suppressor mutation was mapped in malZ. The mutant gene was cloned. It contained a Trp to Cys
exchange at position 292 of the deduced protein sequence. Surprisingly,
the purified mutant enzyme was still unable to hydrolyze maltose as was
the wild type enzyme, while both were able to release glucose from
maltodextrins. However, the mutant enzyme had gained the ability to
transfer dextrinyl moieties to glucose, maltose, and other
maltodextrins. Thus, it had gained an activity associated with
amylomaltase. It was the MalZ292-associated transferase reaction that
allowed the utilization of maltose. In addition, we discovered that
mutant and wild type enzymes alike were highly active as
-cyclodextrinases.
The Escherichia coli maltose system (1, 2) contains two enzymes that are necessary for the utilization of maltose and maltodextrins. Maltotriose, after having been taken up by the high affinity and binding protein-dependent ABC (ATP binding cassette) transport system (3) is recognized by amylomaltase (encoded by malQ)(4) , the reducing end glucose is released and the maltosyl residue is transferred to another maltotriose molecule thus forming maltopentaose (5, 6) . The repetition of this cycle leads to the formation of long maltodextrins and free glucose which, after phosphorylation by glucokinase enters glycolysis. Maltopentaose and longer maltodextrins are recognized by maltodextrin phosphorylase (encoded by malP) (7, 8) which, by phosphorolysis, releases the nonreducing end glucose as glucose 1-phosphate. Thus, the final products of maltodextrin metabolism by these two enzymes are glucose and glucose 1-phosphate.
The
degradation of maltose, the smallest member of maltodextrins, also
requires amylomaltase, and malQ mutants are
Mal. However, amylomaltase does not recognize maltose
as glucosyl donor, only as maltodextrinyl acceptor(6) .
Therefore, in order to metabolize maltose, amylomaltase requires a
maltodextrin primer with the minimal size of maltotriose. Within the
cell, the required maltodextrin primer can originate from the
degradation of glycogen or from the action of an as yet uncharacterized
maltose/maltotriose phosphorylase with glucose and glucose 1-phosphate
as starting material(9) .
There are two more enzymes,
members of the mal regulon, that are not essential for the
metabolism of maltose and small maltodextrins. One is a periplasmic
-amylase encoded by malS(10, 11) . This
enzyme hydrolyzes larger dextrins in the periplasm preferentially to
maltohexaose (12) which can then be transported by the
maltose/maltodextrin transport system. The other is a cytoplasmic
enzyme encoded by malZ(13, 14) whose
function in maltodextrin metabolism is unclear. The enzyme degrades
linear maltodextrins up to a chain length of 7 glucose units but not
maltose. It sequentially cleaves glucose from the reducing end of the
maltodextrin chain. Even though it can hydrolyze maltotriose to glucose
and maltose, malQ mutants cannot grow on maltotriose or
maltotetraose even when malZ is overexpressed(14) .
The endogenous formation of maltotriose required for maltose utilization is also an important aspect of the regulation of the mal system. All mal genes are under the control of MalT, the positive activator of the system(15) . In vitro MalT-dependent mal gene expression requires the presence of maltotriose(16) . Cells growing in the presence of maltose or any other maltodextrin will result in the elevated expression of the mal system even though only maltotriose is the effective internal inducer. We have argued that the increased formation of endogenous maltotriose during exogenous induction by maltodextrins is driven by glucose and glucose 1-phosphate, the degradation products of maltodextrin metabolism. This reaction is most likely catalyzed by maltose/maltotriose phosphorylase, the same enzyme that is required for the maltodextrin primer synthesis mentioned above(9) .
In an
attempt to identify the postulated maltose/maltotriose phosphorylase,
we reasoned that the action of this enzyme should be reversible, and it
should therefore be able to split maltose to glucose and glucose
1-phosphate. Thus, malQ mutants which are Mal should be able to grow again on maltose after a mutational event
resulting in the overproduction of the postulated maltose/maltotriose
phosphorylase. Among the phenotypic revertants in such a selection, we
found one mutation that did not map in malQ. In this paper, we
report the analysis of this second site revertant. We found that the
mutation had occurred in malZ. We cloned and sequenced the
mutant malZ (malZ292). We purified the encoded enzyme
and characterized its enzymatic activity. We present the explanation
for the ability of the mutant enzyme to support growth on maltose by
its acquisition to transfer dextrinyl residues to maltose, followed by
the consecutive hydrolysis of glucose from the transfer product.
Figure 1:
Cloning of malZ292. a, region of the E. coli chromosome around minute 9
of the genetic map that is cotransducible with the malQ suppressor mutation. b, plasmid pRP100 that had been
selected for its ability to complement a phoB::Tn5 mutation and which carried the malZ292 mutation. c and d, subcloning of malZ292 for minimal size.
The letters above the lines show the restriction sites: B, BamHI; M, MluI; P, PstI, Ps, PshAI; S, SacII.
Figure 7:
The formation of linear maltodextrins,
labeled at the reducing end glucosyl residue. The reaction mixture
contained 100 mM maltotriose, 0.4 mM [C]glucose (5 µCi), and 0.5 mg of crude
extract of a MalQ overproducing strain in 250 µl of 50 mM Tris-HCl, pH 7.0. After 1 h, the mixture was chromatographed by
descending paper chromatography on Whatman 3MM with
butanol:ethanol:water (5:3:2) as solvent. The figure represents a
portion of the autoradiogram with the formed radioactive substrates
indicated on the left.
MalZ activity was assayed by following the formation of p-nitrophenol from p-nitrophenyl maltoside (PNG2) as described previously(14) . Units of enzymatic activity are given in micromoles of PNG2 hydrolyzed per min at room temperature.
Figure 2: SDS-polyacrylamide gel electrophoresis of the different fractions in the purification of MalZ and MalZ292. Lane 1, molecular mass standards; lanes 2-5, purification of MalZ292; lanes 6-9, purification of wild type MalZ. Lanes 2 and 6, crude extracts of cells that have undergone heat induction; lanes 3 and 7, guanidinium HCl-insoluble pellet remaining after solubilization and renaturation of inclusion bodies; lanes 4 and 8, renatured protein after solubilization of inclusion bodies; lanes 5 and 9, homogeneous protein after Mono Q ion exchange chromatography. Except for the pure protein, 10 µg of total protein was applied on each lane. The gels consisted of 10% polyacrylamide and were stained with Coomassie Blue.
Figure 3:
Hydrolysis of maltopentaose by wild type
MalZ and MalZ292 in the presence of trace amounts of
[C]glucose or [
C]maltose,
demonstration of the transfer reaction. Unlabeled maltopentaose (10
mM), [
C]glucose (25 µM),
or [
C]maltose (10 µM) was incubated
in 50 mM sodium phosphate buffer, pH 7.0, with 2 µg of
pure protein. The assay volume was 30 µl. Samples of 10 µl were
spotted on TLC plates 2, 10, and 30 min after the addition of the
enzyme. Lanes 1-3, wild type MalZ with
[
C]glucose; lanes 4-6, wild type
MalZ with [
C]maltose. Lanes
14-16, MalZ292 with [
C]glucose; lanes 15-19, MalZ292 with
[
C]maltose. Lanes 7-11, sugar
standards as indicated on the left. The TLC plate was
developed with butanol:ethanol:water (5:3:2). A, chemical
detection by charring with sulfuric acid; B, autoradiography
prior to charring.
Figure 4:
The dextrinyl transfer reaction of
MalZ292. Unlabeled maltose (lanes 1 and 5),
maltotriose (lanes 2 and 6), maltotetraose (lanes
3 and 7), and maltopentaose (lanes 4 and 8) at concentrations corresponding to 20 mM glucosyl
residues were incubated with 10 µM [C]maltose (lanes 1-4) or 20
µM [
C]glucose (lanes
5-8) in 15 µl of 50 mM sodium phosphate, pH
7.0, with 1 µg of pure MalZ292 for 2 min. 10 µl were spotted
onto a TLC plate and developed with butanol:ethanol:water (5:3:2). A, chemical detection by charring with sulfuric acid; B, autoradiography prior to
charring.
When
[C]maltose was used as acceptor, the first
products to appear with maltotriose as donor were
[
C]maltotetraose as well as
[
C]maltotriose. With maltotetraose as donor,
[
C]maltopentaose as well as
[
C]maltotetraose appeared as first products.
This is consistent with the mechanism of transfer onto
[
C]glucose mentioned above. Dextrinyl transfer
onto [
C]maltose at the nonreducing end would
occur after the enzymatic release of the reducing end glucose moiety of
the donor. This first product will thus contain two consecutive
C-labeled glucose residues at the reducing end. This
product itself is a good substrate of MalZ and will lead to the quick
release of [
C]glucose as well as a
C-labeled dextrin that is smaller by one glucose moiety.
With unlabeled pentaose as donor, the results are less clear since the
products formed were not sufficiently separated by the TLC technique.
It is noteworthy to emphasize that the MalZ292 enzyme, aside from its
increased activity as a dextrinyl transferase, still shows the same
activity as the wild type enzyme in its function as a maltodextrin
glucosidase, i.e. in the net formation of glucose from
maltodextrins.
Figure 5:
Hydrolysis of -,
-, and
-cyclodextrin by MalZ and MalZ292. The equivalent of 20 mM glucosyl residues of
-,
-, and
-cyclodextrin in 15
µl of 50 mM sodium phosphate buffer, pH 7.5, was incubated
with 1 µg of pure MalZ enzyme. After 150 min, 10 µl were
spotted onto a TLC plate and developed with butanol:ethanol:water
(5:3:2) followed by chemical detection with charring. Lanes
1-3, controls of glucose, maltose, and maltotriose; lanes 12-15, controls of
-,
-, and
-cyclodextrin as well as pullulan. Lanes 4-6 and 8-10, assays with
-,
-, and
-cyclodextrin; lanes 7 and 11, assays with
pullulan. Lanes 4-7, incubation with wild type MalZ; lanes 8-11, incubation with
MalZ292.
Figure 6:
The
transfer reaction of MalZ292 with -cyclodextrin as substrate and
[
C]glucose as acceptor. 80 µl of 50 mM sodium phosphate buffer, pH 7.0, containing 3 mM
-cyclodextrin and 25 µM glucose was incubated
with 5 µg of pure MalZ292. 10-µl samples were removed at 1, 5,
10, 20, 40, and 80 min (lanes 2-7) and spotted onto a
TLC plate. The following controls were applied: lane 1,
glucose; lane 8, linear maltoheptaose; lane 9, linear
maltohexaose. The plate was developed with 1-propanol:ethyl
acetate:water (3:2:2, v/v/v) followed by chemical detection with
charring in the presence of sulfuric acid. Lanes 10-15,
autoradiogram of lanes 2-7 prior to charring. Lanes
16 and 17, control [
C]glucose and
[
C]maltose. Note that the position of the large
radioactively labeled compounds at the earlier time points is not
identical with
-cyclodextrin, the starting
material.
When the mutant MalZ enzyme was used in this assay, the amount of glucose released was significantly higher with all maltodextrins tested. We interpret this as the consequence of the maltodextrinyl transfer reaction associated with the mutant enzyme. This would result in the transfer of dextrinyl moieties onto maltose and subsequent release of glucose. This reaction is essentially the reason why the malZ292 mutation in a malQ background gives rise to maltose utilization.
These dextrins were synthesized by the
transferase reaction of amylomaltase. Unlabeled maltotriose (100
mM) was incubated with [C]glucose (0.4
mM) and amylomaltase. The products formed were separated by
paper chromatography, and the results are shown in Fig. 7.
According to the established mechanism of amylomaltase
action(6) , these sugars are labeled exclusively in the
reducing end glucose residue. After rechromatography, maltotriose and
maltotetraose were used in the MalZ-dependent hydrolysis reaction. The
results are shown in Fig. 8. We now conclude that from
maltodextrins longer than maltotriose not only glucose but also maltose
can be released to a small extent.
Figure 8:
Maltodextrins hydrolyzed by MalZ. Samples
of 0.08 µCi of maltotriose (lanes 5 and 7) and
maltotetraose (lanes 6 and 8), C-labeled
exclusively in the reducing end glucose residue, were incubated in 10
µl of 50 mM sodium phosphate buffer, pH 7.0, with 1 µg
of wild type MalZ (lanes 5 and 6) or MalZ292 (lanes 7 and 8) for 60 min prior to TLC
chromatography (butanol:ethanol:water (5:3:2) and autoradiography. Lanes 1-4, controls of
C-labeled glucose,
maltose, maltotriose, and maltotetraose.
Surprisingly, a malTmalP::Tn10 mutant (strain RP97) lacking maltodextrin
phosphorylase and (by the polar effect of the Tn10 insertion
on malQ) also amylomaltase and carrying malZ292 as a
chromosomal mutation utilized maltose much better than strain RP98 (malT
malQ::Tn10
malP
) with malZ292 as a chromosomal
mutation. Careful analysis with the purest available maltose (Merck)
revealed that strain RP98 only grew overnight to an A
of about 0.3-0.5. After a long incubation time (between an
additional 10 and 20 h), growth resumed but was due to malQ
reversions. In contrast, strain RP97 (malP::Tn10 malQ malZ292) grew well and was fully
outgrown overnight on the same purest maltose preparation (Merck). In
addition, the sterile filtered spent medium of strain RP98 (malQ::Tn10 malP
malZ292),
harvested at a time when growth had stopped at A
of 0.4, allowed full growth of strain RP97 (malP::Tn10 malQ malZ292). As tested by TLC, the
spent medium still contained large amounts of maltose. This indicates
that endogenously produced maltodextrin needed for the MalZ292-mediated
utilization of maltose will be partially eliminated by maltodextrin
phosphorylase. Apparently less pure maltose preparations contain small
quantities of maltodextrins allowing the MalZ292-mediated utilization
of maltose even in the presence of maltodextrin phosphorylase.
The malZ292 mutation was isolated as a second site Mal revertant from a malQ mutant but obviously not on pure
maltose. We determined the growth rate of the malQ malZ292 mutant on maltose in comparison to a malQ malZ
strain. While the malZ
strain did not
grow at all, we did notice that the malZ292 derivative grew on
maltose at different growth rates depending on the brand of maltose
that was used as carbon source (Table 3). The introduction of a pgm mutation lacking phosphoglucomutase activity, abolished
the difference in the growth rates between the pure and less pure
maltose and allowed reasonable growth rates and final cell densities in
both cases (Table 3). It was somewhat surprising that the rate of
growth on maltose in a malQ malP pgm malZ292 strain (RP93) was
dependent on the copy number of the malZ292 gene, the rate of
growth becoming considerably higher when malZ292 was
plasmid-encoded than when chromosomally encoded.
The expression of the maltose system is controlled by the level of
maltotriose, the endogenous inducer. Obviously, maltotriose can be
produced and degraded by several enzymes. Thus, it is informative to
analyze the state of mal gene expression in several mutants in
the presence and absence of exogenous maltose. Expression was measured
by maltose transport activity (Table 4). The constitutivity of a malQ mutant is somewhat less in the presence of the malZ292 mutation while the presence of maltose in such a
strain increases the expression more than in a malZ strain (SFC1 versus HS3166). As expected, the
introduction of a glgC or a pgm mutation in SFC1
abolishes the constitutive expression, but does not prevent induction
by exogenous maltose.
The finding that MalZ292 as well as the wild type MalZ enzyme is an
effective -cyclodextrinase raised the question whether or not this
activity is of physiological significance. Strain MC4100, our control
strain as a standard laboratory Mal
strain, cannot
grow on
-cyclodextrin.
In this publication we report the mutational alteration (Trp to Cys at position 292 of the polypeptide chain) of MalZ that results in the effective utilization of maltose when present in a mutant that lacks amylomaltase, the key enzyme of maltose utilization in E. coli(1) . MalZ had previously been identified as an enzyme cleaving glucose from the reducing end of maltodextrins with maltotriose as the smallest substrate. The wild type MalZ enzyme is apparently not involved in maltose utilization since mutants lacking malZ still grow normally on maltose and strains harboring the wild type malZ gene but defective in malQ cannot grow on maltose(29) . However, in pgm mutants which still can grow on maltose(27) , the function of the malZ-encoded enzyme becomes apparent. Growth on maltose is strongly reduced. Thus, MalZ increases the ratio of glucose over glucose 1-phosphate, the immediate products of maltose utilization.
The mutant enzyme MalZ292 still exhibits the same activity as the wild type MalZ enzyme. Surprisingly, maltose itself was not hydrolyzed by the mutant enzyme. The only difference that we could detect was the ability of the mutant enzyme to transfer dextrinyl residues originating from maltotriose and larger dextrins onto maltose. This indicates that it is the transfer reaction onto maltose that allows maltose utilization. The transfer reaction observed with the mutant MalZ enzyme is reminiscent of the action of amylomaltase, the gene product of malQ. This enzyme will disproportionate any given maltodextrin longer than maltose into glucose and a series of maltodextrins in such a way that the number of glycosidic linkages will remain constant. Thus, amylomaltase is exclusively a maltodextrin transferase but not a hydrolase. For every glucose released (the apparent hydrolysis reaction), it will form a new glycosidic linkage by producing a longer dextrin.
Even though the hydrolysis and the transfer reaction observed with the mutant MalZ enzyme is formally the same as in amylomaltase, there is still a basic difference between the two enzymes. In the mutant MalZ, hydrolysis of glucose is not obligatorily coupled to the transfer reaction, and net hydrolysis of maltodextrins to glucose and maltose is still the predominant reaction. Nevertheless, with a continuous supply of small amounts of dextrins (maltotriose and larger), maltose can be degraded to glucose by the mutant MalZ enzyme. This does not require the presence of an additional enzyme. In contrast, when maltose is degraded to glucose by the ``normal'' amylomaltase-mediated pathway, aside from the requirement of a maltodextrin primer, the action of maltodextrin phosphorylase, the malP product, is needed to remove the accumulation of longer dextrins (produced by amylomaltase) by producing glucose 1-phosphate that enters glycolysis after phosphoglucomutase-mediated transformation into glucose 6-phosphate.
The properties of the mutant MalZ enzyme again necessitate postulating an endogenous production of maltodextrins that can act as a dextrinyl donor in the MalZ292-mediated utilization of maltose. In the malQ mutant, the major source of these dextrins including maltotriose, the inducer of the system, is clearly glycogen(9, 30) . However, also the pgm or the pgm glgA derivative of the malQ malZ292 strain can still grow on maltose. These strains do not contain detectable glycogen and are not constitutive for the maltose system even though they still can be induced by maltose. Thus, it is obvious that there exists a second pathway for the synthesis of endogenous maltodextrins that do not originate from glycogen. We have previously postulated the existence of a maltose/maltotriose phosphorylase that would produce maltose from glucose and glucose 1-phosphate and additionally maltotriose from maltose and glucose 1-phosphate(9) . Since phosphorylases are reversible, maltose plus phosphate would produce glucose 1-phosphate which then could give rise to the synthesis of small maltodextrins needed for the MalZ292-mediated maltose utilization in the malQ mutant background. The observation that pgm mutants in this background exhibit an increased rate of maltose utilization is consistent with this picture. Glucose 1-phosphate produced from maltose (by the postulated maltose/maltotriose phosphorylase) would not be removed by phosphoglucomutase (forming glucose 6-phosphate followed by glycolysis). A testable prediction would therefore be that malQ pgm mutants when exposed to maltose will have an elevated level on glucose 1-phosphate even in the absence of maltodextrin phosphorylase. Clearly, the postulated maltose/maltotriose phosphorylase cannot be very active. Otherwise, this enzyme alone should give rise to growth on maltose in the absence of any other mal enzymes.
In the course of this
investigation, we observed that the MalZ enzyme, wild type as well as
MalZ292 mutant, was able to hydrolyze -cyclodextrin, but not
-cyclodextrin, and
-cyclodextrin only to a minor extent.
Several cyclo-maltodextrinases have been isolated in the past and
identified as a special type of maltodextrin hydrolase. They exhibit
molecular weights of 66,000-72,000 and differ from
-amylases, to which they exhibit sequence homology (including
their conserved motifs), by their weak activity in hydrolyzing starch.
Generally, they exhibit transglycosylating activity and some of them
are pullulanases hydrolyzing an
-(1
6)
linkage(31, 32, 33) . MalZ, even though
exhibiting
-cyclodextrinase activity, carries latent
transglycosylating activity that becomes prominent only after mutation.
We could not detect any hydrolyzing activity on pullulan consistent
with the claim that E. coli does not contain pullulanase (34) . At present, the physiological role, if any, of the
-cyclodextrinase activity of MalZ is unclear. E. coli is
unable to transport or to grow on
-cyclodextrin.