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INTRODUCTION |
Azotobacter vinelandii is a rod-shaped, Gram-negative
dinitrogen-fixing soil bacterium that under adverse environmental
conditions undergoes a differentiation process that transforms the
vegetative cell into a dormant, desiccation-resistant form (cyst)
(1-9). In the cyst state the modified vegetative cell (central body) is surrounded by a protective capsule consisting of a thin outer layer
(exine) and a thicker inner layer (intine), both rich in proteins,
lipids, and carbohydrates. When the requirements for growth are again
fulfilled, the A. vinelandii cell escapes the cyst capsule
and re-enters the vegetative state.
An important component in the intine and exine layers is alginate, a
1,4-linked linear polysaccharide consisting of
-D-mannuronic acid
(M),1 and its C-5 epimer,
-L-guluronic acid (G). The M- and G-moieties are
distributed as heteropolymeric (MG) and/or homopolymeric (MM or GG)
blocks in the polymers (10-15). The alginates appear to be essential
to the differentiation process, since non-mucoid strains fail to encyst
(16-18). A. vinelandii in addition synthesizes alginates
during vegetative growth. This vegetative capsule seems to be
responsible for bacterial adhesion to surfaces and may function as a
diffusion barrier against O2 or heavy metals (19).
Bacterial alginates are O-acetylated in varying degrees at
O-2 and/or O-3 on mannuronic acid moieties (20-22).
In all studied alginate-producing organisms the epimerization of M to G
is catalyzed by mannuronan C-5-epimerases at the polymer level
(23-29). The A. vinelandii genome encodes a family of seven secreted Ca2+-dependent epimerases (AlgE1-7)
(29) and a Ca2+-independent periplasmic epimerase (AlgG)
(28) similar to the epimerase (AlgG) in Pseudomonas
aeruginosa (25). The AlgE epimerases consist of varying numbers of
two types of structural modules, A and R, of which the A-module alone
is capable of catalyzing epimerization and determining the final
distribution of G-moieties in the produced alginates (30). The
R-modules contain four to seven Ca2+-binding motifs, and
the direct binding of Ca2+ by this module, as well as by
the A-module, has been demonstrated previously (30). The R-modules
stimulate reaction rates and are probably involved in export of the
AlgE proteins (23, 31, 32). By producing a family of alginate
epimerases, A. vinelandii is able to synthesize a variety of
alginates with different physicochemical properties, the exact
biological function of which is not yet clearly understood.
The genomes of alginate-synthesizing organisms encode alginases that
degrade the polymers at specific uronic acid sequences, but most
bacterial lyases display low activity against the native highly
acetylated polymers (33-35). The alginases associated with A. vinelandii are reported to cleave M-rich alginates most
effectively (36-39), even though the periplasmic AlgL enzyme degrades
M-G bonds with similar efficiency as M-M bonds (36). Interestingly, the AlgE7 protein displays both epimerase and lyase activity (29). In 1987 Gacesa (40) proposed that the reaction mechanism of these two enzyme
activities were very similar (Fig. 1).
Both mechanisms include removal of H-5 of the uronic acid, but instead
of a replacement of this proton from the opposite side of the sugar
ring as in epimerization, the alginases catalyze a -elimination of
the 4-O-glycosidic bond, generating unsaturated
4-deoxy-L-erythro-hex-4-enepyranosyluronate moieties at the non-reducing end. Based on stereochemical
considerations, Feingold and Bentley (41) predicted that a similar
reaction mechanism could apply to both M-specific and G-specific
lyases. If these theoretical considerations are correct it seemed
possible that the AlgE7 lyase and epimerase activities originate from
the same active site in the enzyme. In this paper we examine the
relationship between these two reactions mediated by AlgE7, and the
results strongly support the hypothesis that a common site is
responsible for both activities.
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Fig. 1.
The proposed reaction mechanisms for alginate
lyases and epimerases (made on the basis of a figure by Gacesa
(40)). AA1-3 refer to amino acid residues on the enzyme.
The negative charge on the carboxylate anion is believed to be
neutralized by a positively charged amino acid (AA1) in the active site
of the enzyme. Note that the abstraction of the H-5 proton is believed
to occur from below the sugar plane, whereas the replacement occurs
from above. For simplicity the protons are omitted from the sugar
rings.
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EXPERIMENTAL PROCEDURES |
Growth of Bacteria--
Escherichia coli cells were
grown in L-broth with shaking (unless otherwise stated) or on L-agar at
37 °C (42). When relevant the E. coli media were
supplemented with 200 µg/ml ampicillin unless otherwise stated. For
enzyme purification, the E. coli cells were grown in a
medium containing 30 g/liter tryptone, 15 g/liter yeast extract, and 5 g/liter NaCl (3× concentrated L-broth).
Standard Laboratory Methods--
Restriction endonuclease
digestions, ligations, and agarose gel electrophoresis were performed
as described by Sambrook et al. (42). Transformations were
performed according to Chung et al. (43). Plasmids encoding
AlgE6 (29), AlgE7 (29), AlgE7-E1, AlgE1-E7, and AlgE7-D152G were
propagated in E. coli SURE (44), whereas pHH5 (45), encoding
AlgE5, was propagated in JM109 (46). Plasmids were isolated by a midi
kit from Qiagen (for sequencing) or a Wizard miniprep kit from Promega.
DNA sequencing for verification of plasmid constructs was performed by
using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready
Reaction kit (PerkinElmer Life Sciences) and an Applied Biosystems
Model 373 automatic sequencer. Protein concentrations were measured
using the Bio-Rad Coomassie Brilliant Blue-based protein assay using
bovine serum albumin as standard.
Plasmid Constructions and Site-specific Mutagenesis--
AlgE1
was previously shown to consist of two separate catalytic domains, of
which the N-terminal part, AlgE1-1, introduces both G- and MG-blocks
into the alginate substrates (encoded by pHE37) (47). AlgE1-1 has the
same modular structure as AlgE7 (AR1-3), and its epimerization products
have been extensively characterized (47). The expression vectors pHE37
and pBG27 (encodes AlgE7 (29)) were digested by NcoI and
BglII, and the 5'-798 bp of algE1-1 were
substituted with the same A-module encoding part of algE7
and vice versa. The resulting plasmids, pBG70 and pBG71, encode and
express the hydrid enzymes AlgE7-E1 (N-terminal 266 amino acids from
AlgE7) and AlgE1-E7 (N-terminal 266 amino acids from AlgE1), respectively.
For site-specific mutagenesis pBG27 was digested by NcoI and
BglII, and the 5'-798 bp of algE7 were inserted
into the cloning vector plitmus29 (New England Biolabs). The resulting
plasmid, pBG78, was used to mutate the aspartic acid residue at
position 152 in AlgE7A to glycine (D152G) (QuikChangeTM
site-directed mutagenesis kit, Stratagene) using the oligonucleotides E73, 5'-CGCTACGCCTTCGGGCCCCACG-3', and E74,
5'-CGTGGGGCCCGAAGGCGTAGCG-3'. The resulting plasmid, pBS2,
was digested by NcoI and BglII, and the 5'-798 bp
containing the D152G mutation were substituted by the 5'-798 bp of
pBG27, generating pBS4 (expresses the AlgE7-D152G mutant protein).
Alginates Used as Substrates--
Acetylated mannuronan was
isolated from the epimerase negative P. aeruginosa strain
FRD462 (48). A deacetylated (20) preparation of this alginate was used
in all analyses involving mannuronan. For the NMR experiment referred
to in Fig. 6, the mannuronan substrate was degraded to an average
degree of polymerization (DPn) ~ 100 by mild acid
hydrolysis (12) before incubation with AlgE7. Polyalternating alginate
(MG-alginate) was produced in vitro from mannuronan by using
recombinantly produced AlgE4. Since AlgE5 and AlgE6 do not display
lyase activity, this MG-alginate was degraded to a DPn = 30-40
before incubation with these two enzymes (NMR analyses). The highly
acetylated M-rich alginate was isolated from P. aeruginosa
8830 (49), and the acetylated G-rich alginate was isolated from
vegetatively growing A. vinelandii E (26, 50).
Deacetylated versions (20) of these two alginates were also used.
The Macrocystis pyrifera alginate is a commercially available high molecular weight alginate from Sigma. The G-block alginate was isolated from the outer cortex of Laminaria
hyperborea stipes and degraded to DPn = 30-40 by acidic
hydrolysis and fractionation (11) before incubation with AlgE7 and
AlgE7-E1. 5-3H-Labeled M-rich alginate (specific activity
144330 dpm/mg alginate) was prepared by growing P. aeruginosa 8830 (49) in a medium containing
5-3H-labeled glucose (28). The composition of the alginate
substrates used are summarized in Table I.
Preparation of Enzyme Extracts--
E. coli cells
containing the plasmid of interest were grown overnight in 3×
concentrated L-broth supplemented with 400 µg/ml ampicillin. The
cultures were diluted 1:100 in the same medium, and after 3 h of
incubation the production of enzymes were induced by adding
isopropyl--D-thiogalactopyranoside at a concentration of
0.5 mM. The cells were harvested 3-4 h after induction and resuspended in 
of the culture volume in MC buffer (MOPS (pH
6.9), 2.2 mM CaCl2) and then disrupted by ultrasonication. After centrifugation at 27,000 × g
for 30 min, the crude cell extracts were filtered through a 0.2-µm
Millipore filter before purification by ion exchange chromatography.
The crude cell extracts were loaded onto a HiTrap Q Sepharose high
performance column (Amersham Pharmacia Biotech) equilibrated with MC
buffer. All tested proteins were eluted by a continuous NaCl gradient
(0 to 1 M NaCl in MC buffer); AlgE7 was eluted between 36 and 39 mM NaCl, AlgE7-E1 was eluted between 33 and 38 mM NaCl, AlgE5 was eluted between 44 and 48 mM
NaCl, and AlgE6 was eluted between 30 and 35 mM NaCl.
Measurements of Enzymatic Activities by Isotope Assays (Lyase and
Epimerase) and Spectrophotometry (Lyase)--
A radioisotope assay
(29) was used for detection of epimerase and lyase activities in the
crude extracts (AlgE7, AlgE7-E1, AlgE1-E7, AlgE7-D152G, AlgE5, and
AlgE6) and for identification of active enzyme fractions after
purification with ion exchange chromatography (AlgE7, AlgE7-E1, AlgE5,
and AlgE6). The definition of the specific activity of AlgE7 and
AlgE7-E1 (Fig. 2) is complex since two
reactions (epimerization and degradation) run simultaneously, and both
reactions are influenced by changes in the structure of the substrate
(FG increases) as the reactions proceed. The two reactions
cannot be easily quantified separately also because H-5 may be released
twice from the same moieties after epimerization followed by cleavage
at the same site. Measured radioactivity (dpm) in the supernatant (in a
scintillation counter) after incubation and precipitation of alginate
(29) includes, besides released 3H from alginate to water
upon epimerization/degradation, also 3H in unprecipitated
small oligomers produced by the lyase. The specific activities for
AlgE7 and AlgE7-E1 were therefore defined as unprecipitated
radioactivity (dpm/min)/mg of protein. The incubation time used (85 min) in the above calculations was based on reactions that were run to
less than 10% completion. Generally we used samples without added
enzyme as blanks (negative controls) in the experiments, but to get a
relatively accurate estimate of the low activity of the AlgE7-D152G
mutant protein it was found necessary to use the same cells without the
algE7 gene inserted into the plasmid (pTrc99A) as negative
controls. By also using the same amount of added extract for the mutant
and the negative control we were able to design the experiments such
that mutant activities were about 600 dpm, whereas those of the control
extracts were around 50 dpm. Mutant activities also responded linearly
to reductions in the amounts of added extract (values below 600 dpm),
whereas the negative controls remained nearly constant.
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Fig. 2.
SDS-polyacrylamide gel electrophoresis of
purified AlgE7 and AlgE7-E1. Lane 1, crude extract of
AlgE7; lane 3, purified AlgE7, specific activity 1.3 dpm/min/mg of protein × 104; lane 4, crude
extract AlgE7-E1; lane 6, purified AlgE7-E1, specific
activity 1.4 dpm/min/mg of protein × 104. Lanes
2 and 5, molecular mass standard (Bio-Rad). The numbers
on the left refer to the standard (kDa).
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Alginate lyases produce unsaturated
4-deoxy-L-erythro-hex-4-enepyranosyluronate
moieties (
) as a result of degradation of the alginate substrates,
and these unsaturated uronic acids strongly absorb UV light at 230 nm
(51). Therefore, the lyase reaction was measured as a continuous
increase in absorbance at 230 nm at room temperature by adding enzyme
extract to a mixture of alginate (0.91 mg/ml) in 50 mM
Tris-HCl buffer (pH 7.1) and 2 mM CaCl2 (unless
otherwise stated). The total reaction volume was 1.1 ml. Lyase activity
is given as the increase in absorbance units per minute.
Determination of the Dependence of the AlgE7 Lyase Activity on
Divalent Cations, pH, and Ionic Strength--
The lyase activity of
AlgE7 was assayed in the pH range 6.5-8.8. The buffers used were MOPS
(pH 6.5-7.0; pH adjusted with NaOH) and Tris-HCl (pH 7.0-8.8; pH
adjusted with HCl). The pH in each reaction was measured in the cuvette
after the lyase reactions were stopped. Before measurements of the
lyase activity as a function of divalent cations and NaCl
concentrations, the AlgE7 preparation was dialyzed extensively against
20 mM MOPS (pH 6.9) and then concentrated against 20%
polyethylene glycol (Mr = 20 000). For the
substrate specificities and NMR analyses, the enzyme was not dialyzed,
resulting in final NaCl concentrations of ~34 mM
(substrate specificity analyses) and 10 mM (NMR spectroscopy).
Measurements of Epimerase and Lyase Activities by 1H
NMR Spectroscopy--
Samples for characterization by NMR spectroscopy
were incubated in a total volume of 7.2 ml containing the relevant
enzyme, 8 mg of alginate in 50 mM Tris-HCl buffer (pH 7.1),
and 2 mM CaCl2 at 37 °C. When using the
G-block alginate (AlgE7 and AlgE7-E1), the acetylated M-rich P. aeruginosa alginate (AlgE7), and the degraded (in
vitro) MG-alginate (AlgE5 and AlgE6), the reactions were stopped
by adding Na2EDTA to a concentration of 10 mM (removes remaining Ca2+). The samples were
then dialyzed against deionized water (dH2O) before NMR
spectroscopy. The acetylated alginate from P. aeruginosa was, in addition, deacetylated (20) after incubation with AlgE7 but
before NMR. No other reaction mixtures involving AlgE7 and AlgE7-E1
were dialyzed before NMR spectroscopy due to the degradative activity
of these enzymes and, hence, potential loss of small oligosaccharides
through the dialysis membrane. The AlgE7-D152G mutant protein (crude
extract) was incubated (24 h) with mannuronan and the M. pyrifera alginate. The reactions were then stopped and dialyzed
against deionized water (dH2O). The alginates were then
degraded by mild acid hydrolysis (DPn~30-40) (12) before NMR
spectroscopy. All substrates and products were freeze-dried and
dissolved in D2O (10 mg/ml at pD 6.8). The samples were
then analyzed by NMR spectroscopy using a Bruker AM-300 (300 MHz)
spectrometer at 90 °C. The internal standard used was
3-(trimethylsilyl)-propionic-2,2,3,3-d4-acid, Na+ salt (Aldrich).
For the specific NMR experiment referred to in Fig. 6 the AlgE7
reaction on mannuronan was performed in an NMR tube inside the
spectrometer. 12.0 mg of mannuronan (DPn~100) was dissolved
in 400 µl of D2O. 300 µl of this solution was
transferred into a 5-mm NMR sample tube, and CaCl2 in
D2O, Tris-HCl buffer (50 mM, pH 7.0 at
49 °C), and 5 µl of 1%
3-(trimethylsilyl)-propionic-2,2,3,3-d4-acid, Na+ salt
(internal standard) were added to a total volume of 450 µl. A
1H NMR spectrum of this starting material was recorded.
Then 50 µl of the enzyme solution (2.2 mg of the enzyme preparation
in 150 µl of D2O) was added, and continuous NMR recording
was started. The final concentrations in the NMR sample (500 µl) were
18.0 mg/ml mannuronan, 1.32 mg/ml AlgE7 (as total protein), and 8.15 mM Ca2+.
Spectra were recorded on a Bruker DPX 400 spectrometer. To monitor the
progress of the reaction, a series of 270 1H NMR spectra
was recorded successively (T = 49 °C). Spectra were obtained using a spectral width of 3612 Hz, a 32,000 data-block size,
and 64 scans were accumulated. The resulting time interval between two
successive spectra was 8.5 min. No presaturation or other technique to
reduce the HDO signal was used to avoid distortion of neighboring
signals. The peaks were assigned according to Grasdalen et
al. (12, 13) and Heyraud et al. (52).
Due to different reaction conditions, the degradation time for
mannuronan found in the above-described NMR experiment cannot be
directly compared with the incubation time needed for complete degradation given in the substrate specificity curve (see Fig. 4A). Additionally, the time courses of the epimerase and
lyase activities were analyzed by stopping the reaction at different time intervals before completion (terminated by freezing, no dialysis), whereupon the reaction products were analyzed in ordinary NMR experiments (results not shown). The NMR data acquired from integration of these spectra were consistent with the corresponding results shown
in Fig. 6B.
Interpretation of 1H NMR Spectra--
The molar
fraction of the monomers G (FG), M (FM), the
diads (FGG, FMM, FGM,
FMG), and the G-centered triads (FGGG,
FMGM, FGGM, FMGG) were calculated
as described by Grasdalen et al. (12, 13). The average
length of the G-blocks was calculated from NG>1= (FG 
FMGM)/FGGM.
Alginate lyases degrade alginates at specific sequences and produce
unsaturated 4-deoxy-L-erythro-hex-4-enepyranosyluronate moieties () by a -elimination reaction. The characteristic
resonance signals from the reducing ends (Gred and
Mred) and from the unsaturated nonreducing ends, , may
then be identified and their fractions calculated from the NMR spectrum
of the degraded alginate sample (52). Moreover, since the latter
resonance shifts depend on the nearest neighbor moiety, both
M and G can be identified. Thus, when G
MM and/or GGM bonds
are cleaved, the result will be Gred signals from the
reducing end and M signals from the nonreducing end. If instead
MMG and/or MGG bonds are attacked, the corresponding resonance
signals will be Mred and G. From the NMR spectrum, only
the -anomeric reducing end signals can be integrated due to overlap
of the -signals with the unsaturated nonreducing -1-G end
signals. To calculate the total molar fraction of G- and M-moieties at
the reducing ends, the intensities of the -signals are found by the
ratios of the anomeric protons M
/M
2.2 and G/G 0.2 (52). The average degree of polymerization, DPn, was estimated from DPn = [IG-5 + IM-1 + (IGred + IMred) × 2]/[IGred + IMred],
where I denotes the integrated intensities of the indicated
signals in the spectra.
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RESULTS |
Purification and Biochemical Characterization of AlgE7--
The
plasmid pBG27 (29) was used for expression of AlgE7 in E. coli SURE, and the enzyme was purified by ion-exchange
chromatography in a one-step protocol, which was found to be sufficient
for further biochemical characterization (Fig. 2). The band
representing AlgE7 in the SDS-polyacrylamide electrophoresis gel
corresponded to a higher molecular mass than expected based on the
deduced amino acid sequence of the enzyme (105 kDa compared to 90.4 kDa, respectively). Such aberrant migration rates, possibly
A-module-associated (30, 53), have been observed also for other AlgE
proteins (45, 47, 54).
To study the relation between the epimerase and lyase activities of
AlgE7, the response of the lyase to the parameters, known to be
important for epimerization, was analyzed. The experiments showed that
Ca2+ is an absolute requirement for the lyase activity
(Fig. 3A). Maximal activity
was reached at a concentration of 2.5 mM, and it remained
constant up to the highest tested concentration, 18 mM.
Similar to epimerization by AlgE1 (47), AlgE2 (55), and AlgE4 (54),
only Sr2+ could substitute for Ca2+, although
the efficiency of the reaction was reduced by ~80% (Fig.
3B). At a suboptimal calcium concentration (0.5 mM), Mn2+ was inhibitory, whereas
Zn2+ eliminated all activity. Mg2+ on the other
hand had a weak stimulatory effect. The detrimental effect of
Zn2+ was also observed for AlgE1 (47), AlgE4 (54), and for
the periplasmic epimerase AlgG (28). In conclusion, the responses of
the AlgE7 lyase to divalent cations are very similar to that of the
epimerases, strengthening the hypothesis that the same catalytic site
is involved in both enzymatic activities.
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Fig. 3.
Biochemical characterization of the AlgE7
lyase. A, Ca2+ dependence; B,
effects of different divalent cations; C, ionic strength
dependence. Mannuronan was used as substrate.
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To ensure optimal lyase activity in further experiments we also
determined the pH and ionic strength optima. The pH optimum was found
to be between 6.9 and 7.3, with about 85 and 74% activity at pH 6.5 and pH 7.9, respectively (results not shown). The lyase activity of
AlgE7 was inhibited by high NaCl concentrations, as also observed
earlier for AlgE epimerases (47, 54, 55). At 300 mM only
11% of the activity was retained (Fig. 3C). The epimerase
activity of AlgE7 was not followed in these experiments, since the
isotope assay does not distinguish between epimerization and lyase
activity (see "Experimental Procedures"). However, the conditions
found to be optimal for the lyase are very close to those previously
shown by NMR spectroscopy to support good epimerization activity of
AlgE7 (29).
The Substrate Dependence of the AlgE7 Lyase Activity--
The AlgE
epimerases are known to display different activities on different types
of alginates (45, 47, 56), for example by being sensitive to
acetylation (57). Furthermore, since the epimerization reaction appears
to act mostly or exclusively in the M to G direction (54), it follows
that G-block alginates are very poor substrates for the epimerases. If
the epimerase and lyase activities originate from the same site in
AlgE7, one might therefore expect its lyase activity to act poorly on
highly acetylated alginates and substrates with continuous stretches of
G-moieties. To analyze this we used eight structurally different alginate substrates (Table I). The
experiments showed that AlgE7 displayed lyase activity on all tested
alginates, except on the G-block alginate from L. hyperborea
(Fig. 4A). Deacetylated M-rich alginates (mannuronan and alginate from P. aeruginosa 8830)
and the M. pyrifera alginate were found to be the best
substrates (equal maximum lyase activity), and the overall kinetics on
the two M-rich alginates appeared nearly identical. Noteworthy,
however, was that complete degradation of the M. pyrifera
alginate required a longer incubation time with the enzyme than the
M-rich substrates. One possible explanation for this may be the
existence of more unfavorable G-block sequences in this substrate.
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Table I
Alginates used in this study
See "Experimental Procedures" for a further description of the
different alginates.
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Fig. 4.
Substrate specificity analysis of AlgE7.
A, AlgE7 lyase activity on different alginate substrates.
All lyase reactions were monitored until no further increase in
absorbance (A230) was observed (except for the
MG-alginate, see "Results"); B, initial AlgE7 lyase
activities (first 30 min) on selected substrates. M. pyr.,
M. pyrifera; A.v., A. vinelandii;
deac, deacetylated; ac, acetylated.
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The native highly acetylated (d.a. ~ 0.7) M-rich alginate from
P. aeruginosa was a very poor substrate for the lyase
reaction. The maximum activity on the acetylated alginate was only 15%
that of the activity on the corresponding deactylated substrate, and after about 7 h the lyase reaction almost ceased. At this point more than 90% of the glycosidic linkages remained intact.
Correspondingly, the maximum lyase activity on the less acetylated
(d.a. = 0.06) and deacetylated alginates from A. vinelandii was much more similar, although the final ~50% of
the attacked bonds were cleaved faster in the deacetylated
alginate. Note also that the rate of degradation of the deacetylated
A. vinelandii alginate was significantly slower than that of
the three most efficiently degraded alginates. These experiments
therefore highlight the negative effects of acetylation and continuous
stretches of G-moieties for the activity of the AlgE7 lyase, similar to
what one would expect also for the AlgE7 epimerase activity (see above paragraph).
The MG-alginate was degraded much less efficiently than the M-rich
alginates, and the maximum lyase activity was only 17% compared with
the activity on mannuronan. Furthermore, AlgE7 degraded the amount of
mannuronan used within 1.8 h, whereas the same quantity of
MG-alginate was not completely degraded after 52 h.
Mannuronan and the M. pyrifera alginate were degraded with
nearly similar efficiency (Fig. 4A), but a closer analysis
of the kinetics revealed that the initial degradation profiles differed (Fig. 4B). Pure mannuronan was degraded significantly slower
initially and was found to behave similarly to the deacetylated
alginate from A. vinelandii. This suggested to us that
G-moieties in M-rich areas somehow stimulate the lyase reaction,
but that this effect requires a certain sequential distribution of the
G-moieties (Table I). The epimerase activity of AlgE7 might introduce
such favorable sequences in the alginates, explaining the overall
efficient degradation of M-rich substrates.
1H NMR Analyses of Alginates Incubated with
AlgE7--
To obtain more detailed information on the substrate
specificity of the AlgE7 lyase and the interplay between the lyase and epimerase activities, the reaction products from the different substrates were analyzed by NMR spectroscopy. The spectrum of completely degraded mannuronan showed, as expected, that AlgE7 acted as
a combined epimerase and lyase, and the G content in the degraded
alginate after epimerization was 17% (Fig.
5A and Table
II). In the spectrum the signals
from Gred dominate over those from Mred,
whereas the unsaturated nonreducing end signals exclusively are M.
This is in good agreement with previously reported results using an
M-rich (FG ~ 0.05) alginate from P. aeruginosa
as substrate (29). Hence, potential cleavage sites for the AlgE7 lyase
with mannuronan as substrate are GMM and/or GGM, and MMM
and/or MGM sequences, with a clear preference for G at the reducing
end of the cleavage site (i.e. the two first mentioned
bonds). From the signal intensities of the end groups, the average
degree of polymerization (DPn) of the oligomers was calculated
to represent a tetramer, of which 35% of the G-moieties were internal
(Table II).
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Fig. 5.
1H NMR spectra of alginates
before and after incubation with AlgE7, AlgE7-E1, and AlgE5.
A, mannuronan (AlgE7 and AlgE7-E1); B, acetylated
and deacetylated M-rich alginates from P. aeruginosa (AlgE7
only); C, MG-alginate (AlgE7, AlgE7-E1, and AlgE5). In the
different spectra G, M,
Gred, Mred, and denote internal G- and M-moieties, reducing end G- and M-moieties, and
4-deoxy-L-erythro-hex-4-enepyranosyluronate
moieties, respectively. The numbers refer to the position of
the proton in the pyranosyl ring that causes the signal, and the
nonunderlined M and G refer to the neighboring moieties (see
also "Experimental Procedures"). For all substrates except the
MG-alginate, the lyase reactions were allowed to proceed to completion
(no further increase in A230). Since the
specific activity of AlgE7 and AlgE7-E1 is very similar (Fig. 2),
equimolar amounts of enzymes were used in reaction with the
MG-alginate. The NMR data of the integrations of the peaks are
summarized in Table II.
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Table II
Composition and sequence parameters of alginates before (B) and after
treatment with AlgE7, AlgE7-E1 and AlgE5
The corresponding NMR spectra for some of the reactions are presented
in Figs. 5 and 6A.
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The kinetics of the AlgE7 activities could also be followed by carrying
out the reaction inside an NMR tube, allowing spectra to be obtained
continuously as the reaction progressed (Fig.
6A). Visual inspection
indicated that the internal G content (G-1 peak) reached its
maximum relatively early and then slowly decreased, whereas the lyase
activity, on the other hand, continued to degrade the substrate, as
demonstrated by the increase in the end signals. By integration of the
17 spectra in Fig. 6A, a more quantitative relationship
between moieties epimerized, bonds cleaved, and DPn could be
obtained (Fig. 6B). The plot confirmed that the epimerase activity dominated initially and that the AlgE7 epimerase increased the
G content to 26% (the fifth spectrum in Fig. 6A and
Table II). In the same period of time the lyase activity degraded the substrate (DPn = 100) to oligomers with an average size of 11 units (3-4 chain breaks per 26 units epimerized). From this point in
the reaction more G-moieties were consumed in cleavage than were formed
by epimerization, resulting in a slow decrease in FG (Fig.
6B). These data therefore indicate that the oligomer size of
the alginate substrates also influences the balance between epimerization and degradation by AlgE7. In the final spectrum of the
series, the G content had decreased from 26 to 18%, and on average
every fourth bond in the alginate was cleaved by the lyase (DPn ~ 4). This is in very good agreement with the NMR data obtained from
completely degraded mannuronan (Fig. 5A and Table II).
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Fig. 6.
Epimerization and degradation of mannuronan
by AlgE7. A, 1H NMR spectroscopy
demonstrating the interplay between epimerization and degradation of
mannuronan (17 selected spectra) (see Fig. 5 for abbreviations). The
AlgE7 reaction was allowed to proceed in an NMR tube inside the
spectrometer. Since the enzymatic reaction took place in
D20, no resonance signals from H-5 could be
recorded. The direction of the arrow indicates increasing
reaction time (17 spectra); B, units epimerized (represented
by the molar fraction of G moieties, total FG), bonds
cleaved (represented by the molar fraction of -1-M,
F -1-M), and DPn, plotted as a function of
incubation time with the enzyme. The three parameters analyzed were
calculated by integration of the corresponding spectra in A.
To retain the clarity of the plot, the first four spectra in
A were stacked wider than indicated by the time scale
(compare with B).
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The type and intensity of the end signals found in degraded M. pyrifera alginate were similar to those in the mannuronan spectra (the M. pyrifera spectrum is not shown). Hence, the
degradation of this substrate involved the same glycosidic links as
those attacked during the reaction on mannuronan, with the same
apparent preference toward the GMM and/or GGM bonds. The M. pyrifera alginate was degraded to a DPn of about 7 (Table
II), and most of the guluronic acids were found as internal G-moieties (83%). Taking into account the sequential distribution of G-moieties in this substrate, the end products of the lyase activity may be
considered as a mixture of fairly long G-blocks and smaller oligosaccharides with a predominance of M-moieties. This is consistent with the finding that G-blocks are not cleaved by the lyase (Fig. 4A). Even though some newly epimerized moieties are likely
to be converted to units in the lyase reaction (with a concomitant decrease in total FG), the NMR data indicate that the lyase
activity dominated on this particular alginate (Table II). This
observation is interesting in that it clearly shows that the
composition of the substrate can determine whether the enzyme acts as
an epimerase or a lyase. For a possible explanation of this, see
"Discussion."
The influence of acetylation on the lyase and epimerase activities of
AlgE7 was also studied by NMR spectroscopy using the P. aeruginosa M-rich alginate (~5% G) as substrate (Fig.
5B and Table II). The analysis confirmed the inhibitory
effect of the acetyl groups on the lyase activity (DPn = 43),
and the spectra in addition showed also that the epimerase activity was strongly inhibited by these groups (FG = 0.11). In
contrast, a deacetylated version of this alginate was readily degraded
to a DPn of about 7, and the G content in the degraded alginate had increased to 30%. The chemical shift values and intensities of the
lyase signals from the deacetylated M-rich alginate were similar to
those obtained with mannuronan as substrate. The G content and
especially the amount of internal G-moieties in the degraded alginates
were nevertheless significantly higher with the M-rich alginate.
Moreover, the degraded M-rich alginate contained more than twice as
many GG sequences as depolymerized mannuronan. One possible
explanation is to assume that the epimerase has a preference for
introduction of new G- moieties next to a pre-existing one in the
M-rich alginate, leading to the formation of short G-blocks. Since
mannuronan does not contain G-moieties initially, the formation of
G-blocks may be much less efficient on this substrate. This hypothesis
is interesting in view of the previously suggested processive or
preferred attack modes of action of the AlgE4 epimerase (54).
AlgE7 Is Capable of Epimerizing Polyalternating
Alginates--
A strictly polyalternating alginate has only two
possible cleavage sites for lyases, GMG and MGM, and according to
results described above, only the latter of these bonds represents a
potential site for the AlgE7 lyase. Furthermore, it was not expected
that the AlgE7 epimerase could introduce more favorable GGM cleavage sites for the lyase, since it was previously reported that single M-moieties in a GMG sequence were inaccessible to epimerization (45,
47, 58). Thus, one would expect the lyase activity on this substrate to
generate M-moieties at the reducing ends and M-moieties at the
nonreducing ends and that no further epimerization of the substrate
would take place. Surprisingly, however, the spectrum from the MG-
alginate contained both M and G signals in equal amounts
(measured as intensities), whereas the reducing end signals were
exclusively Gred (Fig. 5C and Table II).
Furthermore, the total G content increased from 46 to 62%,
demonstrating that AlgE7 had epimerized M-moieties situated in a GMG
sequence. Thus, cleavable AlgE7 bonds in the MG-alginate after
epimerization were GMG and GGM, where the G at the reducing end
in the latter was introduced by the AlgE7 epimerase. In addition, the
absence of Mred signals illustrated that MGM sequences
are inaccessible for AlgE7 on the polyalternating alginate. This leads
to the conclusions that polyalternating alginates can be epimerized by
AlgE7, that the new alginate structure thereby generated becomes a
substrate for the lyase activity, and that G signals are generated
at the nonreducing end. The latter was not observed with the other
tested substrates. The results therefore suggest that there exists a very complex relation between substrate composition and the possible lyase cleavage sites.
Since the epimerization of the polyalternating alginate was unexpected,
we also tested two other epimerases, AlgE5 and AlgE6, for this
property. The results showed that the G content increased from 46 to 58 and 54%, respectively (Fig. 5C, Table II; results only
shown for AlgE5). This ability is therefore not unique to AlgE7.
The Properties of an Enzyme Hybrid (AlgE7-E1) and a Point Mutant
Protein (AlgE7-D152G) Strongly Indicate That the Same Site Is
Responsible for Both the Epimerase and the Lyase Activities--
We
have recently shown that the A-modules are sufficient for epimerization
(30), and based on the hypothesis that the epimerase and lyase
activities originate from the same catalytic site, we compared the
amino acid sequences of the AlgE7 A-module to those of AlgE1-6 (24,
29). The analysis showed that at least two distinct short motifs in the
N-terminal part are particularly characteristic for AlgE7 (29). This
might indicate that the N-terminal part of the A-module contains
moieties that are needed to display lyase activity, and to test this
hypothesis we substituted the 5' 798 base pairs in algE1-1
(47) with the corresponding sequence from algE7, generating
algE7-E1, and vice versa (algE1-E7). The
resulting two plasmids, pBG70 and pBG71, could then be used for
expression of AlgE7-E1 and AlgE1-E7 in E. coli SURE. The
crude extracts were analyzed with respect to epimerase and lyase
activities (radioisotope assay), and the results showed that AlgE7-E1
displayed strong epimerase and/or lyase activity, whereas the activity
of AlgE1-E7 was 1-2% that of AlgE7-E1. AlgE1-E7 was not studied
further as it was difficult to obtain NMR spectra showing the activity. To elucidate if the active hybrid enzyme displayed both epimerase and
lyase activity, AlgE7-E1 was purified (ion exchange chromatography) and
incubated with different alginates before NMR spectroscopy.
Incubation with mannuronan followed by NMR spectroscopy of the reaction
products showed that AlgE7-E1, similarly to AlgE7, displayed both
epimerase and lyase activity (Fig. 5A). It could therefore
be concluded that the N-terminal 266 amino acids of AlgE7 are
sufficient to convert an epimerase to a combined lyase and epimerase.
The type of the end signals were also identical to those generated by
AlgE7 acting on the same substrate. However, the relative intensities
of the reducing end signals were different, as AlgE7-E1 seemed to have
a relatively higher preference for M-moieties at the reducing end (Fig.
5A). If one assumes that the epimerase activities of AlgE7
and AlgE7-E1 are important for producing good substrates for the lyase
reaction, it was not unexpected that the total G content in the
AlgE7-E1-degraded mannuronan was lower than for AlgE7 and that the
internal FG after complete degradation was much smaller
than the total FG for both enzymes (particularly significant for AlgE7-E1, Table II).
The difference in substrate specificity compared with AlgE7 was also
reflected by the appearance of weak but clearly significant G
signals from reaction products of the M. pyrifera alginate and the G-block alginate (spectra not shown). Since AlgE7-E1 appears to
cleave the alginates more effectively than AlgE7, we expected that the
MG-alginate might also serve as a better substrate for the hybrid
enzyme. This assumption was confirmed by the observed strong intensity
of the G signal relative to M in the corresponding NMR spectrum
(Fig. 5C) and by the lower DPn value generated by
the hybrid enzyme (Table II). Similar to AlgE7, the hybrid enzyme was
also capable of epimerizing M-moieties between two G-moieties.
The experiments described above indicated that at least some of the
N-terminal 266 amino acids in the AlgE7 A-module are of key importance
for the lyase activity but also that the substrate specificity of the
lyase is affected by amino acids located in the C-terminal part. Even
though these experiments were consistent with the hypothesis that the
two enzyme activities originate from the same site in the A-module,
possibly in the N-terminal part, it was not obvious what specific amino
acids would be essential for both activities. No inactive epimerase
point mutants have so far been reported, and we therefore carried out a
computer analysis to try to identify candidate amino acids that might
affect one or both activities. A total of nine A-modules have been
sequenced from the AlgE-type family, but all these sequences share too
much similarity to point to a particularly critical amino acid. This problem can be approached in different ways, but here we decided to
first focus on a comparison of the AlgG and AlgE epimerases (Fig.
7), which share a very low degree of
overall sequence similarity. However, short stretches of similarity
could be detected, and we focused on a region in which there is also
some similarity to a pectate lyase (PelL) and an apparently unrelated
virus protein (GP1). Based on this comparison, only one amino acid
(Asp-152 in AlgE7) was found to be universally present (Fig. 7).
Site-specific mutagenesis was therefore used to mutate
algE7 such that the aspartic acid at position 152 was
changed to glycine. The mutant enzyme was expressed in the same vector
system as AlgE7, and Western blot analyses showed that it was expressed
equally well as the wild type enzyme (not shown). The crude extract was
then subjected to activity analyses by the radioisotope assay (M-rich
alginate, ~5% G), and interestingly, only a very weak activity could
be detected (about 0.2% of the wild type enzyme). Since both the lyase
and epimerase activities are measured by this assay, it seemed as both
activities had been virtually eliminated by this single amino acid
substitution. The result was also confirmed by NMR analyses (mannuronan
substrate), as neither signals from epimerization nor lyase activity
could be detected (NMR spectrum not shown).
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Fig. 7.
Sequence alignment of protein segments found
by BLAST software within the mannuronan C-5-epimerases and other
proteins. The aspartic acid (D) residues conserved in all
sequences are in bold and underlined.
AlgE1-6 Av, mannuronan C-5-epimerases AlgE1 (Q44494), AlgE2
(Q44495), AlgE3 (Q44496), AlgE4 (Q44493), AlgE5 (Q44492), and AlgE6
(Q9ZFH0) from A. vinelandii; AlgE7 Av, mannuronan
C-5-epimerase AlgE7 (Q9ZFG9) from A. vinelandii; AlgY
Av, A-module containing protein AlgY (Q9ZFG8) from A. vinelandii; AlgG Av. mannuronan C-5-epimerase AlgG
(P70805) from A. vinelandii; AlgG Pa, mannuronan
C-5-epimerase AlgG (Q51371) from P. aeruginosa; GP1
Esv, GP1 protein from Ectocarpus silliculosus virus
(Q90190); PelL Ec, pectate lyase from Erwinia
chrysanthemi (Q47473), which belongs to the family 9 of pectate
lyases. a, an identical sequence is present at positions
989-1002 and 987-1000 in AlgE1 Av and AlgE3 Av proteins,
respectively. b, the PelL Ec protein displayed a very low BLAST
score.
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Since there is a complex interplay between the epimerase and lyase
activities, it was formally possible that the epimerase activity alone
had been inactivated by the mutation and that lack of this activity
resulted in an unfavorable substrate (mannuronan) for an intact lyase
activity. To investigate this, we also repeated the NMR experiment by
using the G-containing M. pyrifera alginate as substrate
(NMR spectrum is not shown). No activity was detected, and it is
therefore clear that both activities became inactivated by the point
mutation. Based on this, many of the other experiments reported here
and on previously reported data on the properties of the AlgE
epimerases, we feel that it is very probable that the epimerase and
lyase activities both originate from the same site in the A-module of AlgE7.
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DISCUSSION |
The results reported here strongly suggest that not only are lyase
and epimerase reactions mechanistically related but that they can both
be catalyzed by the same active site in an enzyme. However, despite the
very large number of alginate lyases studied (59), only AlgE7 has been
reported to display this capacity at comparable rates. This obviously
raises the question as to what conditions have to be met for AlgE7 to
act as an epimerase and/or a lyase. The activity patterns observed when
different alginate substrates were used showed that acetylation and
continuous stretches of G-moieties are inhibitory for both reactions
(Fig. 4A and Table II), and a simple explanation for this
could be that the common alginate binding site is incompatible with
such a substrate composition. It is, however, more difficult to
envision what determines whether the enzyme acts as a lyase or an
epimerase on substrates that can be converted by one or both activities
(i.e. mannuronan and the M. pyrifera alginate).
When using pure mannuronan as substrate, the reaction rate is much
faster for the epimerase up to a point where the substrate has become
highly degraded, where after the lyase activity strongly dominates on
the small depolymerized oligomers (DP < 11) (Fig. 6). This
indicates that small oligomers are readily bound by the enzyme but that
for the epimerization pathway to be favored, longer alginates are
needed. This assumption is in good agreement with a recent study that
showed that epimerization by AlgE4 and AlgE2 cannot take place on
mannuronan substrates smaller than 8 and 6 moieties,
respectively,2 whereas AlgE7
is clearly able to degrade to smaller fragments. The same way of
thinking may also explain the strong dominance of the lyase activity on
the M. pyrifera alginate. AlgE7 can obviously bind to this
polymer, but it may be that epimerization is somehow more sterically
hindered by the nature of the substrate. These differences in substrate
selectivity might be explained by assuming different stereochemical
requirements for binding and positioning of the alginate substrate in
the lyase compared with the epimerase reaction (Fig. 1).
Based on results from NMR spectroscopy, potential cleavage sites for
the AlgE7 lyase were GMM and/or GGM, whereas MMM and/or MGM
sequences are less favorable. GMG sequences were also found to be
cleavable (MG-alginates) but with strongly reduced efficiency. The fact
that G-moieties dominated at the reducing end even when mannuronan was
used as substrate highlights the complexity of the interplay between
the lyase and epimerase activities. It probably means that the
initially higher epimerase activity results in the formation of a new
substrate that is more favorable for the lyase than the
substrate provided from the start. Similarly, GMG sequences
appear to be poorly cleavable, but the activity on substrates containing many such triads (like the MG-alginate) is
self-stimulated by introducing cleavable GGM-sequences through
the fill-in capacity of the epimerase activity.
Even if pure mannuronan and the deacetylated M-rich alginate (~5% G)
are very similar substrates, the data obtained from complete degradation differed in that the total G-content (and FGG)
and especially the amount of internal G-moieties was significantly higher in the M-rich alginate (Table II). As a consequence of this, the
average size of the final oligomers was also higher. This may indicate
that the AlgE7 epimerase preferentially attacks M-moieties next to a G
in the alginate chains, as this mode of action (preferred attack)
probably will result in formation of short G-blocks, which in turn
inhibit the AlgE7 lyase from cleavage.
Although there are obviously certain amino acid residues that are
essential for both cleavage and epimerization to take place (like
Asp-152), the experiments reported here indicate that the specificity
of these reactions appears to be affected by many other residues
in the protein. This was clearly illustrated by the reaction products
of the hybrid enzyme AlgE7-E1, which more frequently contained
M-moieties at the reducing ends compared with the products of the
native AlgE7 reaction. The hybrid enzyme also generated stronger G
signals relative to those of M, when the MG-alginate was used as
substrate (Fig. 5C), and the final DPn was lower
(Table II). For epimerization, the previously reported variability in
the epimerization patterns among the AlgE family enzymes support a
similar conclusion (45, 56), and here we also noted that the M. pyrifera substrate strongly favors the lyase reaction, whereas the
same substrate could easily be epimerized from its original 41% G to
66% by AlgE6 (NMR data not shown). These observations all suggest that
the specificity of both the lyase and epimerase activities are the
result of presumably slight differences in the folding structures of
the enzymes, and to understand the nature of these effects, it seems
obvious that a three-dimensional structure of at least one of the
A-modules would be very important.
Another interesting question not addressed here is what biological
significance the dual properties of AlgE7 might have. It seems likely
that this is somehow related to cyst formation, as it is known that
such differentiated cells contain different types of alginates (15),
and it is also known that the expression of the AlgE enzymes are
affected by the conditions that lead to induction of cyst formation
(53).
,
TOP
ABSTRACT
INTRODUCTION
