(Received for publication, July 31, 1995; and in revised form, October 17, 1995)
From the
Potato D-enzyme was purified from recombinant Escherichia
coli, and its action on synthetic amylose (average M of 320,000) was analyzed. D-enzyme treatment
resulted in a decrease in the ability of the amylose to form a blue
complex with iodine. Analysis of the products indicated that the enzyme
catalyzes an intramolecular transglycosylation reaction on amylose to
produce cyclic
-1,4-glucan (cycloamylose). Confirmation of the
cyclic structure was achieved by demonstrating the absence of reducing
and nonreducing ends, resistance to hydrolysis by glucoamylase (an
exoamylase), and by ``time of flight'' mass spectrometry. The
degree of polymerization of cycloamylose products was determined by
time of flight mass spectrometry analysis and by high-performance
anion-exchange chromatography following partial acid hydrolysis of
purified cycloamylose molecules and was found to range from 17 to
several hundred. The yield of cycloamylose increased with time and
reached >95%. D-enzyme did not act upon purified cycloamylose, but
if glucose was added as an acceptor molecule, smaller cyclic and linear
molecules were produced. The mechanism of the cyclization reaction, the
possible role of the enzyme in starch metabolism, and the potential
applications for cycloamylose are discussed.
D-enzyme (disproportionating enzyme or
4--glucanotransferase, EC 2.4.1.25) was first found in potato
tubers by Peat et al.(1) , but has since been found
in many plant tissues(2) . The enzyme is known to catalyze
glucan transfer from one
-1,4-glucan molecule to another or to
glucose. Malto-oligosaccharides have been shown to be effective donors
and malto-oligosaccharides and glucose to serve as acceptors in
vitro. A maltosyl group has been recognized as the major
transferred unit from the donor molecule(3, 4) . It
has therefore been proposed that the enzyme could be involved in starch
breakdown to produce malto-oligosaccharides upon which starch
phosphorylase can act(2, 5) . Both enzymes are found
in plastids(6) , and a similar function has been proposed for Escherichia coli where D-enzyme (amylomaltase) and
maltodextrin phosphorylase are encoded by genes of the malA operon and are involved in exogenous malto-oligosaccharide
utilization(7) . However, in plants, there is a further
possibility that high molecular weight starch might be the real
substrate of the enzyme. In this case, we could consider the
possibility that D-enzyme may change starch molecule structure and
grain architecture by transferring malto-oligosyl units from one chain
to another(8) . It is known that the enzyme can use soluble
starch (4) or amylopectin (2) as the donor molecule,
but it is not known if the enzyme can use high molecular weight starch
as an acceptor molecule. Thus, further investigations of the action of
the enzyme on high molecular weight starch are important in order to
obtain a better understanding of the role of D-enzyme in starch
metabolism.
We reported the purification of D-enzyme from potato tuber extracts and the isolation of a cDNA clone from potato tuber RNA(8) . Northern blot analysis showed that D-enzyme mRNA is present in leaves, stems, roots, and stolons, but is most abundant in developing and mature tubers(8) . D-enzyme mRNA accumulates under circumstances in which starch biosynthesis is most active, similar to that of enzymes of starch synthesis, including ADP-glucose pyrophosphorylase(9) , granule-bound starch synthase(10) , and starch branching enzyme(11) , which may suggest a role for D-enzyme in starch synthesis(8) . Here, we report that D-enzyme does use high molecular weight amylose as the donor or acceptor molecule and catalyzes a novel cyclization reaction.
Figure 1: Effect of D-enzyme on formation of amylose-iodine complex. A reaction mixture (20 ml) containing 40 mg of synthetic amylose AS-320, 50 mM sodium citrate buffer (pH 7.0), 100 mM NaCl, and 136 units of purified D-enzyme from recombinant E. coli was incubated at 30 °C. At the indicated time points, 1 ml of the reaction mixture was removed, boiled for 5 min to stop the reaction, and then centrifuged. Fifty µl of the supernatant was mixed with 50 µl of distilled water and 2 ml of iodine reagent to measure the absorbance at 660 nm, and 200 µl was used for reducing power measurement by a modified Park-Johnson method (see (13) ). Reducing power when all the amylose was broken down to glucose was defined as 100%.
To determine how the decrease in the blue value is caused
by D-enzyme, possible structural changes in the amylose molecule were
analyzed by gel filtration chromatography. As shown in Fig. 2A, the narrow size distribution of amylose AS-320
was changed to a broad distribution during the initial few minutes, but
subsequently, the products showed a narrow size distribution in the low
molecular weight region (average M of 15,000).
Similar results were obtained using other high molecular weight
amyloses (AS-1000 and AS-110) as substrates, in each case yielding
products with a M
of 15,000 (data not shown).
Since no increase in reducing power was detected, the most likely
explanation for this observation is that D-enzyme catalyzes an
intramolecular transglycosylation reaction (cyclization reaction) to
produce cyclic
-1,4-glucans.
Figure 2: Gel filtration chromatography of products of D-enzyme action on amylose AS-320. Two-hundred fifty µl of the supernatant derived from the experiment shown in Fig. 1was analyzed before (A) and after (B) glucoamylase treatment by gel filtration chromatography using a Superose 6 prep grade column plus a Superdex 30 column with a flow rate of 1.0 ml/min. The standard curve for the molecular mass was produced using synthetic amylose with an average molecular mass of 5, 10, 30, 70, 320, or 1000 kDa as a standard.
The possibility that D-enzyme
could have catalyzed the formation of cyclic -1,4-glucans was
examined by glucoamylase treatment. Glucoamylase is an exo-type amylase
and hydrolyzes both
-1,4- and
-1,6-linkages in starch to
produce glucose from the nonreducing end of the substrate. Thus, linear
or branched glucans are completely broken down to glucose by
glucoamylase. However, glucan with cyclic structure should be resistant
to glucoamylase. The samples shown in Fig. 2A were
treated with glucoamylase, and the resultant glucoamylase-resistant
glucan was precipitated with ethanol and then analyzed with the same
gel filtration column (Fig. 2B). The peak of intact
amylose AS-320 (0 min) was completely hydrolyzed by glucoamylase, but
molecules produced by the action of Denzyme were resistant to
glucoamylase. The amount of glucoamylase-resistant molecules and their
average molecular weights are shown in Fig. 3. The yield of
glucoamylase-resistant molecules increased to its maximum level
(>95%) within 30 min and then remained constant. The average
molecular weight of glucoamylase-resistant molecules estimated by gel
filtration was initially
70,000, but decreased with time to
15,000. The presence of these glucoamylase-resistant molecules
strongly suggested that D-enzyme had catalyzed the cyclization of
amylose AS-320 and produced cyclic
-1,4-glucans.
Figure 3: Yield and average molecular weight of glucoamylase-resistant molecules produced by D-enzyme. The yield of glucoamylase-resistant molecules was measured as described under ``Experimental Procedures.'' The average molecular weight of glucoamylase-resistant molecules was estimated by gel filtration chromatography using synthetic amylose with different average molecular masses as a standard (see Fig. 2B).
Figure 4:
HPAEC analysis of products of D-enzyme
action on amylose AS-320. The products of D-enzyme treatment (18 h) of
amylose AS-320 were analyzed by HPAEC using the conditions described
under ``Experimental Procedures.'' Fifty µg of glucan
before any treatment (A), after glucoamylase treatment (B), and after -amylase treatment (C) was
analyzed. Short-chain amylose was mixed with glucose and
malto-oligosaccharides and used as an
-1,4-glucan marker (D). Positions where
-,
-, and
-cyclodextrins
were eluted are indicated by arrows. Numbers above and beside
peaks (G1, G5, etc.) indicate the DPs of the
products. The amylase treatments were carried out at pH 5.5 and 40
°C. The amounts of glucoamylase and
-amylase used were 50 and
10 units/mg of glucan, respectively.
The glucoamylase-resistant products were
next purified from glucose and then size-fractionated by gel filtration
chromatography. The putative cyclic -1,4-glucans in each fraction
were precipitated with ethanol and then lyophilized. Quantitation of
reducing and nonreducing ends was carried out, but none were detected
(data not shown), consistent with the proposed cyclic structure.
Further evidence for the cyclic nature of the products of D-enzyme
action was obtained by partial acid hydrolysis. The gel filtration
fractions that contained the lower molecular weight
glucoamylase-resistant glucans were next separated by HPAEC (Fig. 5A). Peaks G-J were purified by further
HPAEC and then partially hydrolyzed with 0.1 N HCl. Products
from peak G hydrolysis eluted as linear molecules with a DP of
1-23 (Fig. 5B). These products were hydrolyzed to
glucose by glucoamylase (data not shown). The largest product of
partial acid hydrolysis, with a DP of 23 (G23), is assumed to have
resulted from hydrolysis of one glucosidic linkage of the peak G
molecule. Similar results were obtained for all other peaks, except
that the DP of the largest linear product was 1 unit larger for each
successive peak (Fig. 5, C-E). The retention time
of each peak was less than that of the largest linear product.
Figure 5: Partial acid hydrolysis of glucoamylase-resistant molecules. Glucoamylase-resistant molecules were size-fractionated by gel filtration chromatography as described under ``Experimental Procedures,'' and the fractions containing the smallest glucans were analyzed by HPAEC (A). Glucoamylase-resistant peaks G-J were purified by HPAEC and partially hydrolyzed as described under ``Experimental Procedures.'' The elution profiles of each purified peak before (upper trace) and after (lower trace) partial acid hydrolysis are shown (B-E). Numbers above peaks (G1, G5, etc.) indicate the DPs of the products, with the largest product indicated by an arrow.
Final
confirmation of the cyclic nature of the glucoamylase-resistant
products of D-enzyme action and determination of their molecular masses
were obtained by TOF-MS, which can determine the molecular mass not
only of single molecules, but also of several molecules in a mixture. A
glucan with a DP of n in any non-cyclic structure should have
a molecular mass of 162n + 18, whereas a glucan with a DP
of n in a cyclic structure should have a mass of
162n. The fraction containing the smallest
glucoamylase-resistant molecules (Fig. 5A) was
subjected to TOF-MS (Fig. 6). Several peaks were obtained in the
mass spectrometry spectrum, and the molecular masses agreed with the
theoretical values for cyclic glucans with DPs of 17 and greater, but
not with those for linear glucans. From this and previous results, we
conclude that the smallest glucoamylase-resistant molecule (peak A) is
cyclic -1,4-glucan with a DP of 17 (cG17) and that the consecutive
peaks (B, C, D, etc.) are cyclic glucans with DPs of 1 unit longer for
each successive peak (cG18, cG19, cG20, etc.). Therefore, our results
show that D-enzyme catalyzes an intramolecular transglycosylation
reaction on amylose to produce cyclic
-1,4-glucans (cycloamylose)
with DPs ranging from a minimum of 17 (peak A) to a few hundred (Fig. 3).
Figure 6: TOF-MS analysis of glucoamylase-resistant molecules. Size-fractionated glucoamylase-resistant molecules containing the smallest glucans (shown in Fig. 5A) were subjected to TOF-MS analysis. Numbers above each peak indicate the molecular mass of the glucan + 23 (sodium ion).
Figure 7:
Analysis of action of D-enzyme on cyclic
-1,4-glucan in presence and absence of acceptor molecule.
Size-fractionated cyclic
-1,4-glucan (1 mg) with an average
molecular mass of 30 kDa was treated with D-enzyme (1 unit) in the
absence of glucose (A and F) or in the presence of
glucose at a weight ratio (glucose/cyclic glucan) of 0.0005 (B and G), 0.005 (C and H), 0.05 (D and I), or 0.5 (E and J). This
corresponds to glucose concentrations of 0.1-110 mM. The
reaction mixtures were incubated at 30 °C for 18 h and then boiled
to terminate the reaction. The products (100 µg) were analyzed by
HPAEC before (A-E) and after (F-J)
glucoamylase treatment.
D-enzyme has previously been shown to catalyze a
transglycosylation reaction on malto-oligosaccharides, and the action
of this enzyme on small oligosaccharides has been extensively analyzed (3, 4) . However, little is known about the action of
this enzyme on amylose or amylopectin, although it has been
demonstrated that malto-oligosaccharide units (a maltose unit is
preferred) can be transferred from starch to
glucose(2, 4) . The present results clearly
demonstrate novel activities of Denzyme. First, it can catalyze an
intramolecular transglycosylation reaction on high molecular weight
amylose to produce cyclic -1,4-glucans (cycloamylose) with a DP
ranging from 17 to several hundred. Second, amylose can serve as donor
and acceptor. Third, very long
-1,4-glucan units can be
transferred by the enzyme. Fourth, D-enzyme catalyzes the
transglycosidic linearization of cycloamylose when an acceptor is
present. These activities of D-enzyme are explained in Fig. 8.
The reaction starts with D-enzyme attacking an
-1,4-linkage. The
enzyme then transfers the newly formed reducing end of the substrate
either to the nonreducing end of a separate linear acceptor molecule or
glucose (the intermolecular transglycosylation or disproportionation
reaction) or to its own nonreducing end (the intramolecular
transglycosylation or cyclization reaction). The reversibility of these
reactions allows high molecular weight cyclic molecules to be
linearized again by transglycosylation and lower molecular weight
cyclic molecules to be produced subsequently (Fig. 8).
Apparently, the equilibrium tends toward the formation of cycloamylose
with a M
of
15,000 (DP of 90), as shown in Fig. 3.
Figure 8:
Diagrammatic representations of action of
D-enzyme on amylose and cycloamylose. Lines and circles indicate -1,4-glucan chains, where the relative length
represents their relative DP. Ø, glucosyl residue at reducing
end. Reaction 1 is the disproportionation reaction
(intermolecular transglycosylation). Reaction 2 is the
cyclization reaction (intramolecular transglycosylation). Reaction
3 is the transglycosidic linearization reaction (intermolecular
transglycosylation).
Cyclodextrin glucanotransferase also catalyzes
cyclization and disproportionation reactions on
-1,4-glucans(15) . The transglycosidic linearization of
cyclodextrin in the presence of a suitable acceptor has also been
demonstrated (the ``coupling reaction'')(15) . In all
these respects, D-enzyme and cyclodextrin glucanotransferase seem to
catalyze the same reaction, but the major difference is the DP of the
cyclic
-1,4-glucans produced. Cyclodextrin glucanotransferase
produces cyclodextrins with DPs of 6, 7, or 8. Larger cyclodextrins
with DPs of 9-13 have been reported, but only in trace
amounts(16) . D-enzyme produces cycloamylose with DPs ranging
from 17 to several hundred. This observation suggests that there may be
fundamental differences between cyclodextrins and cycloamylose and the
enzymes that act upon them.
Cyclic -1,4-glucans can potentially
adopt antiparallel double helix, single helix, or nonhelical
conformations. Cyclodextrins are known to adopt a nonhelical
structure(17) . Energy calculations of cyclic
-1,4-glucans
in the nonhelical conformation indicated that cyclodextrin with a DP of
6 has the minimum energy and that as cyclodextrin size increases, so
does the energy(18) . The observation that cyclodextrins with
DPs of 6 and 7 are the major products of cyclodextrin
glucanotransferase activity agrees well with such
calculations(19) . It has been reported that linear amylose can
occur in a double-stranded or single-stranded helix
conformation(20) . Fig. 9shows that either conformation
in linear amylose could potentially allow cyclization to form
cycloamylose in either configuration. The Monte Carlo simulation
suggested that the antiparallel double helix structure is the most
likely conformation of amylose in solution(21) , and
crystallographic analysis has shown that linear amylose with a DP of 6
adopts a left-handed antiparallel double helix
conformation(22, 23) . It can be seen how such a
conformation would readily allow cyclization by D-enzyme since donor
and acceptor sites of amylose are juxtaposed (Fig. 9A)
and so may be readily accommodated by the active site of the enzyme.
Energy minimization calculations suggest that cycloamylose in the
antiparallel double helix conformation is more stable than in the
single helix configuration. (
)We therefore favor the view
that cycloamylose adopts the antiparallel double helix structure, but
confirmation awaits structural analysis.
Figure 9:
Diagrammatic representations of
cyclization of amylose in antiparallel double (A) and single (B) helix configurations. Ø, glucosyl residue at
reducing end. Open and closed arrowheads indicate the
sites attacked by D-enzyme (donor site) and those to which the
-1,4-glucan chain will be transferred (acceptor site),
respectively.
The function of D-enzyme in
plants remains unknown. Preliminary results ()showed that
D-enzyme can also catalyze intramolecular transglycosylation reactions
on amylopectin in vitro. A role for D-enzyme in starch
breakdown can be considered in which linear or cyclic glucans are
produced as substrates for hydrolytic or phosphorolytic enzymes. We
have not yet been able to detect cycloamylose in vivo, which
could be explained if it has a short half-life. However, cycloamylose
may not be produced in vivo if the ratio of acceptor (e.g. glucose) to amylose is high (Fig. 7), but we have no
information on such a ratio in vivo. Alternatively, D-enzyme
could modify starch structure through its glucanotransferase activity.
The function may be revealed when mutants lacking D-enzyme can be
obtained.
Due to the high efficiency of cyclization of amylose by D-enzyme in vitro and the production of recombinant D-enzyme in E. coli, the large-scale production of cycloamylose is feasible. Preliminary experiments have shown that cycloamylose has several interesting properties. It is nonreducing, is highly soluble in cold water, and can form inclusion complexes with several inorganic and organic compounds (data not shown). Cycloamylose may have different dimensions and tertiary structure than cyclodextrins, so that different specificities for guest molecules can be anticipated. Therefore, there is great potential for the exploitation of cycloamylose in chemical, pharmaceutical, and food industries to safely achieve the solubilization, increased stability, sequestration, or altered reactivity of molecules with which it can form inclusion complexes.