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INTRODUCTION |
Alginates are linear copolymers of
-D-mannuronic
acid (M)1 and its C-5-epimer,
-L-guluronic acid (G). The relative amounts as well as
the distribution of these two residues vary greatly between alginates
from different species of both brown algae (1-4) and bacteria (5, 6).
Biologically, alginate appears to serve a variety of functions. Brown
algae maintain rigidity and flexibility through alginates of various
sequential structures constituting different tissues of the plants (1).
In Azotobacter vinelandii, alginates are major constituents
of the vegetative capsule (7) and of the rigid and
desiccation-resistant walls of metabolically dormant cysts. This
resting stage may be entered in nitrogen-free medium containing certain
carbon sources, such as
-hydroxybutyrate (8). Alginate synthesized
by the opportunistic pathogen Pseudomonas aeruginosa appears
to have multiple roles in pathogenesis: as an immunomodulator (9), as a
virulence factor by protecting the bacterium against phagocytosis (10),
and in the formation of biofilms that contribute to antibiotic
resistance (11) and stable attachment to cell surfaces (12). A role for
alginate in the phytopathogenesis of certain pseudomonades has also
been proposed (13).
The parameters that form the basis for the range of properties and
hence the commercial value of alginates are the chain length and the
fraction and sequential distribution of M and G residues in the
polymer. An alginate rich in G blocks (stretches of consecutive G
residues) converts into a gel by cross-linking with divalent ions. Such
gelling properties are highly appreciated in the food, pharmaceutical,
dental, textile, and paper industries (14) and render this type of
alginate a good material for immobilization of biocatalysts (15, 16).
Alginic acid gels, formed by lowering of pH, may broaden the
application range of alginates at conditions that require such
conditions, e.g. in the development of drug delivery systems
(17, 18). In this context, alginates with a preponderance of MG blocks
(stretches of alternating M and G residues) may be a valuable resource
due to the high acid solubilities displayed by this type of polymer
(3). Alginates with a high mannuronic acid content have acquired
considerable biomedical interest due to its antitumor (19) and cytokine
production stimulatory (20) activities.
Mannuronan C-5-epimerase was first discovered in a culture medium of
A. vinelandii (21) and was found to catalyze the
Ca2+-dependent epimerization of M into G at the
polymer level (22). This reaction represented the first example of an
epimerase acting on a polymeric substrate (23). In the early
investigations, the enzyme was reported to introduce single G residues
or homopolymeric blocks of G, depending on the concentration of calcium
ions in the growth medium (22) or in the incubation mixture (24). Screening of an A. vinelandii gene library has, however,
proved the existence of a family of seven mannuronan C-5-epimerase
genes, algE1, algE2, algE3,
algE4, and algE5 (25) and algE6 and
algE7 (53) six of which are clustered in the genome. The
activities of the protein products of these genes are
Ca2+-dependent, and they contain repeats of two
types of structural units, designated A and R. The 150-amino acid R
module is present in one (AlgE4) to seven (AlgE3) copies. Each R module
furthermore contains four to six repeating sequences corresponding to
putative Ca2+-binding motifs. The function of the 385-amino
acid A module, present in one or two copies, is unknown at present (25,
26). Both modules are highly conserved at the amino acid sequence level among the members of the protein family, indicating duplication and
rearrangement events during evolution (25). Due to the variability in
the number of A and R repeats, the deduced molecular masses of the AlgE
epimerases vary in the range of 57.7-191 kDa. Also encoded in the
A. vinelandii genome is a Ca2+-independent
mannuronan C-5-epimerase, AlgG (27), which does not share any
significant overall amino acid sequence homology with AlgE1-5 and
whose epimerization product is yet unknown.
Based on the enzymes studied so far (AlgE2 and AlgE4), it is clear that
each epimerase catalyzes the production of alginates with distinct
monomer distribution patterns (25, 26). In this paper, a more detailed
study of the mechanistic and biochemical properties of AlgE4 is
reported. This enzyme predominantly produces an alginate with long
stretches of alternating MG sequences. The kinetics of formation of
this sequence pattern could not be accounted for by a random attack
model and therefore led us to suggest alternative modes of action of
the epimerase.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids
pHH4 (25), a previously published derivative of the commercially
available expression vector pTrc99A, was used to express algE4 in the Escherichia coli strain JM105
(28).
Expression and Preparation of a Crude AlgE4 Fraction to Be Used
for Purification
A typical purification scheme involved 4 liters of 3×
concentrated LB medium (29), supplemented with 0.5 mg/ml ampicillin. One-liter cultures were inoculated to 1% from an overnight culture of
JM105(pHH4) in the same medium. After 2.5 h of incubation at 37 °C with shaking, algE4 was induced with
isopropyl-
-D-thiogalactopyranoside at a concentration of
0.25 mM. AlgE4 production was allowed for a maximum of
3 h following induction. The cells were then harvested, resuspended in 1/10 volume of 10 mM MOPS, pH 6.8, and
ultrasonicated. The broken cells were centrifuged at 16,300 × g for 30 min, and the resulting supernatant was filtered
through a sterile Millex-GV membrane (0.22-µm pore size, Millipore)
prior to activity measurements and further purification. This crude
extract was designated Fraction A.
Purification of AlgE4
Ion Exchange Chromatography--
Fraction A was loaded (100 ml/run) onto a HiLoad 26/10 Q-Sepharose HP column (Pharmacia)
equilibrated with 10 mM MOPS, pH 6.8. AlgE4 was eluted with
a stepwise 0-1 M NaCl gradient (in 10 mM MOPS,
pH 6.8), and the enzyme was eluted between 0.35 and 0.45 M
NaCl. Fractions containing mannuronan C-5-epimerase activity were
pooled and designated Fraction B.
Native Polyacrylamide Gel Electrophoresis Using Model 491 Prep
Cell (Bio-Rad)--
136 mg of protein in an 1 mg/ml solution,
corresponding to Fraction B, was concentrated to 11 mg/ml on a 5 ml
HiTrap Q-Sepharose HP column (Pharmacia). This solution (10 ml) was
dialyzed against Tris/glycine electrode running buffer in a
Slide-a-Lyzer dialysis cassette (Pierce) and further concentrated to 36 mg/ml within the slide. To one half (1 ml) of this sample, an equal
volume of sample buffer (without glycerol) was added. The sample was then loaded onto the stacking gel surface, and electrophoresis was run
at 4 °C at an initial voltage of 250 V and at a final voltage of 400 V. The elution rate was varied within the range of 0.3-1 ml/min.
Separating (8%) and stacking gels were 10 and 3.5 cm in height,
respectively. Gel, sample, and running buffers were made according to
Ornstein (30) and Davis (31) and as described by the manufacturer.
Eluted fractions containing AlgE4 activity were pooled and concentrated
as before application onto the Prep Cell, except that the dialysis step
was performed against 10 mM MOPS, pH 6.8. The resulting,
highly purified extract contained 9.6 mg/ml in a 1.5-ml solution and
was designated Fraction C. This preparation was used for the
biochemical characterizations and for the studies of the kinetics and
the mechanism of action of AlgE4. The purified enzyme was stored frozen
at
20 °C.
AlgE4 Assays
The activity of AlgE4 was assayed either by radioisotope
measurements or by nmr spectroscopy. In the radioisotope assay, which is based on the release of 3H from alginate to water upon
epimerization (32), a [5-3H]alginate originating from
P. aeruginosa was used (27). [5-3H]glucose was
added to bacteria growing on agar plates, followed by scraping off of
cells and exopolymer after a further 3-day incubation. After
resuspension in 0.9% NaCl, centrifugation, and sterile filtration, the
labeled alginate was deacetylated in 0.1 M NaOH,
neutralized, and dialyzed against water. Finally, the polymer was
ethanol precipitated, extensively washed, and freeze-dried. The
specific activity of this substrate was 1.2 × 105
dpm/mg. Except where otherwise stated, assays were carried out with 0.1 µg of protein and 0.1 mg (0.77 mM)
[5-3H]alginate in 10 mM MOPS, pH 6.8, 1.5 mM CaCl2, for 30 min at 37 °C, and in a
total volume of 0.6 ml. The reactions were terminated by addition of 30 µl of 2.5 M NaCl and 0.8 ml of isopropanol. The alginate
was precipitated at
50 °C for 30 min and centrifuged at
16,300 × g for 30 min. The enzyme activities were then
quantified by determining the amount of released 3H in the
supernatant using a scintillation counter. All assays were performed in duplicate.
For determination of the degree of epimerization and residue sequence
parameters of the epimerized alginate, 1H nmr (4) and
13C nmr (2) were carried out. The required amounts of
substrate per sample were 7.5 and 40 mg, respectively. All four diad
combinations, i.e. FMG, FGM,
FMM, and FGG, and triad sequences were
determined for various extents of conversion. Substrates with an M
content of about 96% were obtained from P. aeruginosa (5).
The alginates were deacetylated (5) and partially degraded by mild
hydrolysis (33) to a degree of polymerization
(DPn) of 20-40, prior to epimerization. Except
where otherwise stated, epimerization reactions were carried out with
10 µg of enzyme/mg alginate in 10 mM MOPS, pH 6.8, 1.5 mM Ca2+, and in a total volume of 6 ml at
37 °C. Reaction times are described in figure legends. Reactions
were terminated by chelation of Ca2+ by adding
Na2EDTA (0.5 M, pH 8.0) to 9 mM.
Extensive dialysis of samples against distilled water was performed
before preparation for nmr spectroscopy (33).
Biochemical Characterization of AlgE4
The effect of pH on the activity of purified AlgE4 was
investigated using buffers with buffering capacities covering different pH ranges (34). The buffers used were MES (pH 4.15-5.6), PIPES (pH
6.2-7.3), HEPES (pH 7.5-8.2), and CAPS (pH 9.5-10.5). Each buffer
compound was used at a 10 mM concentration, and the pH values were adjusted with NaOH or HCl. Activity was measured as dpm/µg protein, and characterization results were expressed as percentages of maximum activity. All measurements were performed in duplicate.
AlgE4 Kinetics
Apparent kinetic constants were estimated from the direct linear
plot of Cornish-Bowden and Eisenthal (35). 0.1 µg of enzyme was
incubated for 5-10 min at 37 °C in 10 mM MOPS, pH 6.8;
0.4, 1.5, or 5 mM CaCl2; and 12-150
µM alginate (sugar residues). All assays, including
controls with no enzyme, were performed in triplicate.
To investigate the reversibility of the enzyme reaction, the unlabeled
M-rich P. aeruginosa alginate was used as substrate, as for
the nmr studies. Due to the high activity of the enzyme on this
substrate, incorporation of tritium from labeled water into M residues
(indicating reversibility) could easily be monitored. One might also
have used a G-rich substrate (epimerized in tritiated water) as a
starting material and followed the release of tritium from G residues
into unlabeled water. However, the interpretation of such an experiment
would be difficult, because the epimerization reaction (M to G), which
does not reach a clear end point (see "Results"), would also
contribute to tritium release. 20 mg of alginate was epimerized with
200 µg of AlgE4 (10 µg enzyme/mg alginate) for 4 h, in the
presence of 3H2O (total activity of 2.3 × 1010 dpm) and under conditions otherwise identical to those
used in the nmr studies. Also included in the experiment was a control sample with no enzyme. After precipitation of the alginate as described
for the radioisotope assay procedure, the samples were completely
hydrolyzed and prepared for ion exchange chromatography (36).
Separation of sugar monomers was performed with 0.5-2 M
acetic acid at a flow rate of 2.4 ml/min (36). The total carbohydrate content in the eluted fractions was determined using the
phenol-sulfuric reaction (37). The concentration of uronic acids
corresponding to an optical density of 0.1 at 485 nm is 15.5 µg/ml
for mannuronic acid and 9.2 µg/ml for guluronic acid (38).
Protein Quantification and Sequencing
SDS-polyacrylamide gel electrophoresis, using a 7.5% gel, was
performed according to Laemmli (39), and the gel was stained using
SYPROTM Orange Stain (Bio-Rad). Molecular masses were
determined relative to mobilities of protein standards.
For N-terminal sequence analyses, proteins were blotted onto a
polyvinylidene difluoride membrane (Immobilon), Coomassie stained, and
analyzed on an Applied Biosystems 477A amino acid sequencer. For amino
acid composition analysis, a protein sample of known A280 was lyophilized and hydrolyzed to its component
amino acids in 6 M HCl at 110 °C for 24 h in
vacuo. Quantitative amino acid composition analysis was determined
using an Applied Biosystems model 421 automatic amino acid analyzer.
The extinction coefficient calculated from these data was used in
spectroscopic determinations of AlgE4 concentrations. Alternatively,
the AlgE4 concentration was determined according to Bradford (40) using
bovine serum albumin as standard. The two methods gave results
differing less than 5%.
Mathematical Modelling of the Epimerization Mechanism
The "random attack" model, described under "Results,"
was simulated by a Monte Carlo scheme (41) that takes into account the
inaccessible M residues flanking G residues. This was done by using an
ensemble of alginate chains with a degree of polymerization (DPn) of 30 and simulating the epimerization by
randomly selecting residues in the ensemble, disallowing epimerization
of previously epimerized residues or their flanking residues. In the
"processive" model, AlgE4 is assumed to randomly attack the
mannuronan substrate and epimerize a given residue. Without
dissociation of the enzyme-substrate complex, it then epimerizes a new
residue by relocating itself two residues along the polysaccharide
chain. As a result, a strictly alternating MGMG sequence is introduced.
The processivity refers to the average number of converted residues per
attack and is given by 1/(1
p), where p
is the probability of relocation along the chain and (1
p) is the probability of dissociation of the enzyme-substrate complex. In terms of triad fractions it can be shown
that for AlgE4, p = (FGMG(t)
FGMG(t=0))/(FMGM(t)
FMGM(t=0)), where t is
the time at which the epimerization reaction is stopped. The model was
implemented using a Monte Carlo scheme (41, 42) based on an ensemble of
alginate chains with a degree of polymerization
(DPn) of 30, as in the random attack model. It
was additionally assumed that the enzyme relocates only in one
direction along the substrate. Furthermore, the number of
enzyme-substrate complexes was continuously updated in accordance with
the changing substrate concentration (defined as the concentration of M
residues not neighboring G residues). In the "preferred attack" model, the enzyme is assumed to preferentially epimerize residues proximal to M residues that have a neighboring G. To accomplish this,
it binds to intrapolymer GMMM segments with a larger binding constant
than to MMMM segments. Consequently, the fraction of GMGM becomes
higher than that of MMGM following epimerization. Because AlgE4 is
assumed not to epimerize M residues neighboring G residues, the number
of sequences that can act as substrates is limited. The polymer is thus
considered to consist of two different types of substrates, GMMM and
MMMM, competing for the pool of enzymes. Although the copolymer nature
of the substrate indicates that several types of substrates coexist in
the alginate chains, this two-substrate approximation is the simplest
extension over the one-substrate model that can be realized. The
preferred attack model was implemented using a Monte Carlo scheme (43,
44) on the basis of alginate chains with a degree of polymerization (DPn) of 30, as above.
The simulation models are available upon request.
 |
RESULTS |
Expression and Purification of AlgE4--
The plasmid pHH4 was
used to overexpress AlgE4 intracellularly in E. coli, and
the enzyme was finally purified from this source. The level of
expression was relatively low (Fig. 1,
lane 2), but following ion exchange chromatography the
specific activity increased 6-fold (Table
I, Fraction B) relative to that of the
crude extract (Fraction A). Interestingly, the four main bands present
in Fraction B (Fig. 1, lane 3), corresponding to molecular
masses of 79, 74, 68, and 58 kDa, were absent in control cells lacking
the algE4 gene. The proteins in these bands were all
subjected to N-terminal sequence analysis, and the results showed that
the sequences in all four cases were identical to the deduced sequence
of AlgE4. Thus, it appears that more than one form of the enzyme is
being made in E. coli.

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Fig. 1.
SDS-polyacrylamide gel electrophoresis of the
purification of recombinant AlgE4. Lane 1, standards;
lane 2, crude extract (16 µg applied); lane 3,
following Q-Sepharose HP ion exchange chromatography (3 µg applied);
lane 4, following native polyacrylamide gel electrophoresis
(1 µg applied).
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Table I
Purification of mannuronan C-5-epimerase AlgE4
The table summarizes the processing of 4 liters of culture medium.
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A further 2-fold increase in specific activity was obtained by native
polyacrylamide gel electrophoresis (Table I, Fraction C), and the
epimerase activity was found to correlate with the band corresponding
to a 79-kDa protein (Fig. 1, lane 4). This is a
significantly higher molecular mass than to be expected on the basis of
the deduced sequence (57.7 kDa). In this purification step the 58-kDa
protein was also purified to near homogeneity, and the corresponding
fraction was found not to display epimerase activity. The nature of
this and other forms different from the 79-kDa protein has not been
further studied.
The extinction coefficient for AlgE4, which was used in specific
activity measurements, was determined to be 0.89. The concentration of
AlgE4 could then be calculated as A280/0.89 mg/ml.
Biochemical Characterization of AlgE4--
All characterization
experiments were carried out on the basis of Fig.
2a, which shows that AlgE4
activity is linear for long incubation times in the presence of excess
substrate. The temperature optimum is close to 37 °C, but the enzyme
activity is maintained at a broad range of temperatures. Preincubation
of the enzyme affects the activity only at temperatures as high as
50-60 °C (Fig. 2b).

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Fig. 2.
Biochemical characterization of AlgE4.
a, AlgE4 activity profile. See "Experimental Procedures"
for standard assay conditions. b, effect of incubation
temperature on the activity of AlgE4. The enzyme was preincubated at
the different temperatures indicated, for 0, 30, and 60 min before
addition of the substrate and continuation of incubation for another 30 min at the indicated temperatures. c, Ca2+
optimum of AlgE4. The enzyme was incubated at different concentrations
of CaCl2 and alginate, as indicated. d, effects
of divalent cations on AlgE4 activity. In reactions with multiple ions,
the concentration refers to that of each individual ion.
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The pH activity curve showed that AlgE4 has an optimum between 6.5 and
7.0 and that the enzyme activity is very sensitive to alkaline pH
values. At pH values above 8 virtually all activity was lost (data not
shown). The AlgE4 activity was found to be nearly optimal in the
absence of NaCl, but additions up to 0.1 M did not reduce
the activity much. As the concentration increased beyond this value,
activity dropped, and at 0.5 M NaCl less than 10% of the
activity was retained (data not shown).
Because the activities of the epimerases have long been known to depend
on Ca2+, this parameter was analyzed in more detail (Fig.
2c). Optimal AlgE4 activity was obtained at around 1-3
mM Ca2+ at a substrate concentration (sugar
residues) of 0.5 mM or higher. At lower concentrations of
substrate, the optimal Ca2+ concentration was somewhat
reduced. The negative effects of adding more Ca2+ were
moderate. Experiments were carried out to assure that no substrate
inhibition was occurring, to an upper limit of 1.5 mM. Chelation of Ca2+ with Na2EDTA at equimolar
concentrations (1 mM) resulted in a complete inhibition of
activity, whereas a reduction of the Na2EDTA concentration
to half (0.5 mM) had a much less pronounced effect. Interestingly, the epimerase activity of AlgE4 preincubated with 1 mM Na2EDTA could be restored by the addition of
a molar excess of Ca2+ (data not shown).
The effects of divalent cations other than Ca2+ on an
A. vinelandii mannuronan C-5-epimerase have been
investigated previously (24). The exact identity of this enzyme is,
however, unknown. The data in Fig. 2d demonstrate that
Sr2+ could substitute for Ca2+ with an
efficiency of about 30%. In contrast, Mg2+,
Mn2+, Ba2+, and Zn2+ had no
stimulatory effect on the epimerization reaction in the absence of
Ca2+. At suboptimal concentrations of Ca2+ (0.4 mM), the addition of an equimolar amount of
Sr2+, Mg2+, or Mn2+ stimulated the
activity, whereas Ba2+ and Zn2+ had the
opposite effect (data not shown). At optimal Ca2+
concentrations, on the other hand, all ions tested (except
Mg2+) were inhibitory (Fig. 2d). The inhibitory
effect was particularly strong with Zn2+.
Specificity of the Epimerization Reaction--
In the early
studies of the mannuronan C-5-epimerase reaction, it was observed that
an enzyme fraction from A. vinelandii gave rise to different
epimerization patterns in the alginate, dependent on the
Ca2+ concentration used during incubation (22). A possible
interpretation of this is that the G distribution pattern in the
reaction product of a given enzyme depends on the Ca2+
concentration or that more than one enzyme was present in the A. vinelandii fraction. Each enzyme might then be affected
differently by the variations in the Ca2+ concentration
(45). Fig. 3a shows that the
AlgE4-catalyzed reaction led to the formation of almost exclusively MG
blocks under all Ca2+ concentrations tested. It therefore
appeared that the enzyme is very specific with respect to the sequence
distribution of G residues in its reaction product, and the lack of
homopolymeric G blocks indicates that the alginate formed by AlgE4 is
of a non-gel-forming type (46).

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Fig. 3.
Sequence distribution pattern generated by
AlgE4, monitored by 1H nmr spectroscopy of epimerized
alginate. Reactions contained 10 (a) or 40 µg
(b) protein/mg alginate, and incubations were carried out
for 20 h at the Ca2+ concentration(s) indicated.
Underlined M and G denote signals from M and G
residues, respectively, whereas letters not underlined denote
neighboring residues in the polymer chain. Numbers describe which H in
the hexose is causing the signal. The area of each peak is proportional
to all respective monads, diads, or triads in the substrate.
Calculation of monad, diad, and triad fractions are based on the
following relations (33): FG + FM = 1, FG = FGG + FGM, FM = FMM + FMG, FGG = FGGM + FGGG, FMM = FMMG + FMMM, FMG = FMGM + FMGG, and FGM = FGMM + FGMG.
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During the studies described above we sometimes noticed that under
certain nonstandard conditions a small but significant amount of GGG
and MGG triads were introduced in the alginate as a result of the AlgE4
activity. To investigate this further we incubated the substrate in the
presence of a large excess of enzyme and analyzed the epimerization
pattern by nmr as a function of time. Under these conditions, a
significant amount of GG diads were formed (Fig. 3b),
however at much lower rates and accumulating over longer periods of
time than MG diads. We also studied the effect of the Ca2+
concentration on the accumulation of GG diads. These experiments showed
that FGG increased with increasing Ca2+
concentration, when other parameters were kept constant (data not shown).
Kinetics of Epimerization by AlgE4--
The determination of
kinetic constants for an enzyme such as AlgE4 is complicated by the
fact that the substrate changes throughout the reaction. Classical
Michaelis-Menten kinetics therefore does not apply. However, the
reaction proceeded linearly at a wide range of substrate
concentrations. Furthermore, no substrate inhibition was observed at
very high concentrations. The Vmax and
kcat values could therefore be determined and
were found to be 14.8 µmol min
1 mg
1
protein and 14 s
1, respectively. For determination of the
apparent Km value, the specific activity of the
substrate represented a technical limitation. The labeled substrate was
prepared by growing cells on tritiated glucose, which limits the
possibilities of preparing substrates with specific activities high
enough to assay enzyme activity at the generally preferred lower
concentration limit of 0.2 Km. The
Cornish-Bowden-Eisenthal direct linear plot analysis was nevertheless
performed down to the manageable lower limit of 0.67 Km. This yielded a Vmax value
of 14 µmol min
1 mg
1 protein, which is in
good agreement with that obtained by simply assaying activity at high
substrate concentrations. An apparent Km value of 18 µM (sugar residues) was read from this plot.
A proposed mechanism of epimerization of a uronate residue involves
three steps: the neutralization of the negative charge, the abstraction
of the proton at C5, and finally, the replacement of the abstracted
proton by a proton donor and a concomitant flipping of the
4C1-configurated M residues into
1C4-configurated G residues (47). Water is
shown to function as such a donor (23, 48), and on this basis we
investigated the degree of reversibility of the epimerization reaction
by incubating nonradioactive alginate with AlgE4 in the presence of
tritiated water. By hydrolyzing the epimerized polymer, followed by
separation of the two types of uronic acids, the radioisotope could
then be specifically located. The results in Fig.
4 show that the activity of the G
residues was seven times that of the M residues, indicating that the
reaction predominantly proceeds in the M to G direction. Moreover, the
labeled M residues might not necessarily originate from reversal of the
reaction. Due to the proposed mechanism of epimerization (47), it seems
possible that the enzyme to some extent exchanges protons without
completing the epimerization reaction.

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Fig. 4.
Ion exchange chromatography of completely
hydrolyzed AlgE4-epimerized and unepimerized alginate. The
alginate was incubated in the presence of tritiated water, so that
3H was incorporated into the sugar monomers upon
epimerization. Elution was performed in 12-ml fractions. The first and
second peaks correspond to G and M monomers, respectively (38). ×,
radioactivity of monomers in epimerized alginate, in dpm/ml; ,
concentration of monomers in epimerized alginate, in mg/ml; ,
radioactivity of monomers in unepimerized alginate (control sample), in
dpm/ml; , concentration of monomers in unepimerized
alginate (control sample), in mg/ml.
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The Mechanism of the Epimerization Process--
The fact that
AlgE4 introduces an alternating sequence of M and G residues into its
substrate raises the question of how such a pattern is created. One
possibility is that the enzyme after each epimerization event leaves
its substrate and then randomly selects a new M residue from any of the
polymer molecules in the solution (random attack model) (49). An
important exception to this random pattern of attack would be that M
residues flanking pre-existing G residues would not be selected.
Experimentally, we used nmr spectroscopy to evaluate this model by
following the fractional distribution of sugar sequence triads in the
reaction product as more and more G residues were being introduced by
the activity of the enzyme (Fig. 5). The
peaks in the resulting spectra were integrated, and the triads thus
determined were plotted as a function of total G content
(FG) (Fig. 6). The triads
shown in Fig. 6 (MMG and GMG) are among those that most significantly will contribute to a test of the validity of the random attack model.
It is intuitively obvious that at low degrees of conversion each random
attack would mostly lead to the formation of an MMG triad, and one
would therefore expect the fraction of this sequence to increase as G
residues accumulate in the substrate. The experimental data in Fig. 6,
however, show that this was not the case. In fact, the fraction of MMG
triads (FMMG) remained almost constant throughout the
entire process. Furthermore, in the random attack model one would
naturally expect the triad GMG to accumulate slowly initially, whereas
experimental data indicate a near linear accumulation from the start. A
mathematical simulation of the random attack model illustrates the
disagreement with the experimental data (Fig. 6). We therefore conclude
that the random attack model does not adequately describe the course of
the epimerization process. Instead we propose two other models; the
processive and the preferred attack models, both of which under certain
parameter settings are in agreement with the experimental data (see
Fig. 6 and under "Discussion").

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Fig. 5.
Sequence distribution pattern produced by
AlgE4, monitored by 13C nmr spectroscopy of epimerized
alginate. Reactions were incubated for 0 (FG = 0.04),
5 (FG = 0.15), 14 (FG = 0.21), and 20 min
(FG = 0.31) and represent a selection of the reaction
mechanism analysis data (see Fig. 6). The Ca2+
concentration was 1.5 mM. For spectrum denotations, see the
legend to Fig. 3.
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Fig. 6.
Triad fractions versus
degree of conversion for epimerization of alginate by AlgE4.
Reactions were terminated within a time period of 5-120 min. , ,
experimentally determined triad fractions FMMG and
FGMG, respectively; Solid lines, long
dashed lines, and short dashed lines show calculated
triad fractions for the random attack model, the processive model with
processivity = 10, and the preferred attack model using a
KGMMM/KMMMM (ratio of binding constants) = 25, respectively. For model descriptions, see under "Discussion." The
statistical variation with regard to interpretation of the nmr spectra
was smaller than the symbol areas.
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DISCUSSION |
Purification of AlgE4 to homogeneity by conventional techniques
was complicated by the existence of apparently differently sized AlgE4
molecules in the sample following ion exchange chromatography. Proteolysis to various degrees may have occurred at the crude extract
level. Proteolytic removal of essential amino acid sequences, with or
without a concomitant change in conformation of the truncated molecules, could explain the lack of mannuronan C-5-epimerase activity
displayed by at least one AlgE4 form. An alternative and perhaps more
probable explanation is that the differences in migration rates (under
both denaturing and nondenaturing conditions) are based solely on
conformational heterogeneity. AlgE4 (at least recombinant from E. coli) may exist as a pool of conformationally heterogeneous
molecules, not all of which are active. Multiple bands in a denaturing
gel could then be due to various degrees of denaturation, leading to
differential binding of SDS by the various protein conformations (50).
Suboptimal binding of SDS may further be reflected in the abnormally
high apparent molecular masses of all but the 58-kDa AlgE4 variant.
This protein may have been completely denatured and hence has bound SDS
unrestrictedly. A further indication of conformational heterogeneity of
AlgE4 is the resolution of the AlgE4 pool by hydrophobic interaction chromatography, which we observed in a recent experiment (data not shown).
The ability of Sr2+ to substitute for Ca2+ in
stimulation of epimerase activity may be ascribed to the fact that
these ions have similar ionic radii (51). The slightly stimulatory
effect of Sr2+ in the absence of Ca2+, is thus
not very surprising. The inhibitory action of Sr2+,
Mn2+, Ba2+, and Zn2+ at optimal
Ca2+ concentrations may likewise be explained in terms of
competitive binding of enzyme or of substrate by these cations.
Alginate has a particularly strong affinity for Ba2+ (52),
possibly explaining the relatively strong inhibitory action of this
cation. The negligible effect of Mg2+ at optimal
concentrations of Ca2+ could likewise be due to the very
low affinity of alginate for this type of ion (52). The detrimental
effect of Zn2+ has also been observed for the
Ca2+-independent A.vinelandii mannuronan
C-5-epimerase AlgG (27), possibly indicating an alternative function of
this ion on the action of mannuronan C-5-epimerases in general.
The sequence distribution pattern produced by AlgE4 is mainly
alternating with respect to M and G residues. GG blocks may, however,
be introduced as a consequence of elevated Ca2+ levels in
combination with excessive enzyme concentrations and prolonged
incubation times. This shows that the enzyme is not totally specific
under all conditions.
The analysis of the mode of action of the AlgE4 epimerase clearly
abolished the random attack model. Actually, the end product of such an
attack would not be a strictly alternating structure, because random
attack eventually would yield a substantial fraction of MGMMGM
sequences, which would remain inaccessible to further epimerization. We
therefore propose that the enzyme either slides along the alginate
chain during the epimerization process (processive model) (49) or
leaves its substrate after each single epimerization event and then
preferentially attacks M residues with G residues located as the next
nearest neighbor (preferred attack model) (44). The simulation results
prove that both the processive and preferred attack models agree better
with the experimentally determined triad fractions than the random
attack mode (Fig. 6). With the assumption that 10 residues on average
are converted per attack, the processive model is in reasonable
agreement with the experimental data (Fig. 6). Likewise, a good
agreement with experimental data is obtained when assuming that the
enzyme binds GMMM sequences over MMMM sequences by a factor of 25. Both
the processive and the preferred attack models offer possible
explanations for the introduced sequence patterns and are valid also
for the potentially reversible reaction. However, evidence to
discriminate between the two models is lacking at present. If the
processive model turns out to be true, it would be particularly
interesting, because it would represent the first known example, to our
knowledge, of an enzyme with a mechanism such as this on a
polysaccharide substrate. Further experimental work to clarify these
issues, in addition to the obvious need for structure determination by x-ray crystallography, could involve the use of substrates of various
and well defined degrees of polymerization and various fractions of
alternating sequences.