(Received for publication, November 26, 1996, and in revised form, March 31, 1997)
From the Departments of Crystal structure analysis of Pseudomonas
fluorescens subsp. cellulosa xylanase A (XYLA)
indicated that the enzyme contained a single calcium binding site that
did not exhibit structural features typical of the EF-hand motif.
Isothermal titration calorimetry revealed that XYLA binds calcium with
a Ka of 4.9 × 104
M Endo- In general, glycosyl hydrolases are unusually resistant to proteolytic
attack and thermal inactivation (7). Although calcium ions often play
an important role in conferring structural stability on proteins, only
two cellulases, a hybrid Bacillus glucanase and CelD from
Clostridium thermocellum, have been shown to bind to
Ca2+ and to both Ca2+ and Zn2+
ions, respectively (8, 9). High concentrations of calcium enhanced the
thermostability of both enzymes and decreased the Km
of CelD for 4-nitrophenyl- Recently, the three-dimensional structures of the catalytic domain of
four Family 10 enzymes have been solved (11-14). They all consist of
an ( The objective of this report is to determine the role of the calcium
binding domain in XYLA. The data presented show that occupation of the
calcium binding loop with its ligand protected the enzyme from thermal
inactivation, thermal unfolding, and proteolytic attack. A mutant of
XYLA in which the key residues of the calcium binding domain were
replaced by alanine exhibited thermal stability similar to that of XYLA
complexed with Ca2+ ions; however, the xylanase variant was
susceptible to cleavage by chymotrypsin. The role of the calcium
binding domain in vivo and the possible mechanism by which
the domain evolved are discussed.
Mutated forms of xynA
(encodes the catalytic domain of XYLA) were prepared as described by
Charnock et al. (16). The primers used to generate XYLA Recombinant strains of Escherichia coli
expressing XYLA or its derivatives were cultured as in (17).
Periplasmic fractions of the cells were prepared in the presence of 25 mM EDTA to complex both free and XYLA-associated calcium.
The EDTA was then removed by dialysis against 100 volumes of 10 mM Tris/HCl buffer, pH 8.0, at 4 °C. XYLA was then
purified by anion-exchange chromatography (17). The purified enzymes
were dialyzed against 3 × 1,000 volumes of 50 mM
Tris/HCl buffer, pH 7.5, containing 10 g/liter Chelex 100 (Bio-Rad) to
remove any remaining Ca2+. All buffers used in the
subsequent analysis of the purified enzymes were treated with Chelex
100 (50 g/liter) following the manufacturer's instructions.
XYLA activity was determined
using oat spelt xylan, 4-nitrophenyl- Proteins were incubated with
Native and mutant forms of XYLA (80 µg/ml) were incubated with 1 mM CaCl2 or 1 mM EDTA for 15 min at a range of temperatures, placed on
ice, and aliquots were assayed for residual MUCase activity at
37 °C. Purified proteins were also incubated at 57 °C in the presence of 1 mM EDTA or a range of CaCl2
concentrations. Samples were withdrawn at regular time intervals and
residual MUCase activity assayed as above.
The binding of calcium to native and
mutant forms of XYLA was assessed qualitatively and quantitatively. In
the qualitative assay 10 µg of the purified proteins were subjected
to nondenaturing gel electrophoresis as described previously (22). The
electrophoresed proteins were electroblotted onto nitrocellulose
membranes (Hybond-C; Amersham International). After electrophoretic
transfer the membrane was washed and incubated with 45Ca as
described previously (23). The membrane was then washed with distilled
water and autoradiographed.
Isothermal titration calorimetry (ITC) was used to assess
quantitatively the binding of calcium to XYLA. ITC measurements were
made at 25 °C following standard procedures (24, 25) using a
Microcal Omega titration calorimeter. Proteins were dialyzed extensively against 50 mM Tris/HCl buffer, pH 7.5, containing 100 mM NaCl, and the dialysis buffer was used
for protein heat of dilution controls. During a titration experiment
the protein sample (5-7 mg/ml), stirred (400 rpm) in a 1.3963-ml
reaction cell, was injected successively with 10-µl aliquots of 8 mM CaCl2.
Prior to DSC
measurements proteins were dialyzed extensively against 20 mM MOPS buffer, pH 7.5, containing either 1 mM
CaCl2 or 1 mM EDTA. DSC experiments were
performed by standard procedures (26) using a Microcal MC-2
ultrasensitive differential scanning calorimeter. Measurements were
made with a scan rate of 60 °C/h over a 35-75 °C range, using
sample concentrations of 1 mg/ml. Normalized excess heat capacity data
were then analyzed using Microcal ORIGIN software.
CD spectra were
recorded at 20 °C in a Jobin-Yvon CD6 spectropolarimeter using
quartz cells of 0.01-cm path length. Proteins (1 mg/ml) were dialyzed
as described for the DSC experiments. Spectra were collected before and
after samples had been incubated at 57 °C for 30 min. Each spectrum
was accumulated from at least three scans between 190 and 250 nm and
was corrected for residual protein concentration from the
A280 value.
Sequences between the highly conserved
motif ENXMK at the NH2 terminus and
KXXY at the COOH terminus of the catalytic domains of 23 of
the 28 Family 10 xylanases (the five sequences not compared did not
contain these highly conserved motifs) were aligned using the CLUSTAL W
version 1.6 multiple alignment program (27). Initially, this alignment
comprised 410 amino acid positions but was later reduced to 394 on
removal of the positions corresponding to extended loop 7. Parsimony
analyses were carried out using the PROTPARS routine in the PHYLIP
3.57c Phylogeny inference package
(http://evolution.genetics.washington.edu). To evaluate the robustness
of the inferred trees, 1,000 bootstrap resamplings of the data were
made using SEQBOOT prior to parasimony analysis. A consensus tree was
produced using CONSENSE. Both the SEQBOOT and CONSENSE routines are
included as part of the PHYLIP 3.57c package.
Crystallographic analysis of XYLA in
the presence of 200 mM CaCl2 showed that the
crystals generated contained Ca2+. The topology of the
putative calcium binding domain of the enzyme is shown in Fig.
1. To confirm that XYLA binds to calcium, the purified
enzyme was subjected to nondenaturing PAGE, blotted onto nitrocellulose
membrane, and then probed with 45Ca. The data presented in
Fig. 2A showed that the xylanase existed in
two forms, presumably the monomer and dimer, which migrated differently
on nondenaturing PAGE and confirmed that XYLA bound to calcium (Fig.
2B). The divalent ion did not appear to influence the
oligomeric state of the enzyme (Fig. 2C). To determine the affinity of the xylanase for calcium and the stoichiometry of binding,
the enzyme-ligand interaction was monitored using ITC. Fig.
3A shows a typical calorimetric titration of
XYLA with Ca2+ at 25 °C. After correction for
appropriate heats of dilution the data were analyzed using Microcal
ORIGIN software. The normalized results are shown in Fig. 3B
and give the association constant (Ka) of 4.9 × 104 M
The geometry of the
putative calcium binding domain of XYLA suggests that Asp-256 (O XYLA and XYLA Table I.
Activity of native and mutant forms of XYLA in the presence and absence
of calcium
Biological and Nutritional
Sciences and ** Agricultural and Environmental Sciences,
Department of Cellular Physiology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 and a stoichiometry consistent with one
calcium binding site per molecule of enzyme. Occupancy of the calcium
binding domain with its ligand protected XYLA from proteinase and
thermal inactivation and increased the melting temperature of the
enzyme from 60.8 to 66.5 °C. However, the addition of calcium or
EDTA did not influence the catalytic activity of the xylanase.
Replacement of the calcium binding domain, which is located within loop
7 of XYLA, with the corresponding short loop from Cex (a
Cellulomonas fimi xylanase/exoglucanase), did not
significantly alter the biochemical properties of the enzyme. These
data suggest that the primary function of the calcium binding domain is
to increase the stability of the enzyme against thermal unfolding and
proteolytic attack. To understand further the nature of the calcium
binding domain of XYLA, four variants of the xylanase, D256A, N261A,
D262A, and XYLA
, in which Asp-256, Asn-261, and Asp-262 had all been
changed to alanine, were constructed. These mutated enzymes did not
show any significant binding to Ca2+, indicating that
Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of
XYLA for the divalent cation. In the presence or absence of calcium,
XYLA
exhibited thermal stability similar to that of the native
enzyme bound to Ca2+ ions, although the variant was
sensitive to proteinase inactivation. The role of the calcium binding
domain in vivo and the possible mechanism by which the
domain evolved are discussed.
1,4-xylanases (xylanases; EC 3.2.1.8) catalyze the
cleavage of internal
-1,4 glycosidic linkages in the backbone of
xylans (1, 2). Using hydrophobic cluster analysis, the catalytic
domains of glycosyl hydrolases have been classified into 57 different
enzyme families (3). Members of each family are thought to have evolved
from a common ancestral sequence. Xylanases belong to either Family 10 or 11 (4). Recently, hydrophobic cluster analysis has shown that
several enzyme families have common folds, suggesting that they evolved
from a single sequence (5). Support for the concept of a common
evolutionary link among some glycosyl hydrolase families is provided by
the conservation of the three-dimensional structure of enzymes from
different families, particularly in the vicinity of the active site,
and the observation that glycosidases belonging to a specific clan
cleave glycosidic bonds by the same mechanism (5, 6).
-cellobioside (9, 10).
/
)8-fold barrel structure in which two conserved glutamates function as the catalytic nucleophile and acid/base catalytic residues, respectively. Xylanase A
(XYLA)1 from Pseudomonas
fluorescens subsp. cellulosa is a modular enzyme comprising an NH2-terminal cellulose binding domain linked
to a COOH-terminal catalytic domain. The catalytic domain is unique within Family 10 enzymes as it is the only xylanase described to date
which contains a calcium binding site (located in loop 7) (11), and it
is the only xylanase from either mesophilic or thermophilic
microorganisms which has been shown to be sensitive to proteinases
(15). The function(s) of the calcium binding domain in XYLA and the
structural basis for the enzyme's sensitivity to proteinases remain to
be elucidated.
Mutagenesis of xynA
,
XYLA/Cex, D256A, D262A, and N261A were: 5
-CGATTGGTGTAAGCGGCACTGGAATTACCGGCATAGGGATTAT-3
, 5
-GTCCAGGCCGGCGGCATCGGACGGTGTACGCAAACGCACATC-3
,
5
-CTGGAATTACCGGCATAGGGATTAT-3
, 5
-CGATTGGTGTAAGCGTTACTGGAAT-3
, and
5
-TTGGTGTAATCGGCACTGGAATTACC-3
, respectively. The
nucleotides that introduced the appropriate mutations into
xynA
are in bold.
-cellobioside, and
4-methylumbelliferyl-
-cellobioside (MUC) as described previously
(16). Assays were preformed in 50 mM Tris/HCl buffer, pH
7.5, at 37 °C in the presence of the indicated concentrations of
CaCl2 or EDTA. The cleavage of xylohexaose and the identity
of the products released were evaluated by high performance liquid
chromatography analysis (18). Protein concentration was determined by
A280 nm using a molar extinction coefficient of
58,100 M
1 cm
1 (19) and by the
method of Sedmak and Grossberg (20).
-chymotrypsin in the ratio of 1:10 (w/w) in the presence of either 1 mM EDTA or 0-10 mM CaCl2 at
37 °C. At regular time intervals samples were withdrawn and assayed
for MUCase activity. To monitor production of the proteolytic cleavage
products, XYLA was incubated with
-chymotrypsin in the ratio of 5:1
(w/w) at 37 °C in the presence of 20 mM
CaCl2 or EDTA. Aliquots were removed at each time point
between 0 and 20 min, heated to 100 °C in the presence of 2% SDS,
and then subjected to SDS-PAGE (21). The NH2-terminal
sequence of the major proteolytic product generated (29 kDa) was
determined as described previously (17). To determine the precise size
of this peptide, the proteolytic reaction was terminated by the
addition of phenylmethylsulfonyl fluoride (1 mM) rather
than SDS , and the sample was dialyzed extensively against distilled
water and then subjected to electrospray ionization mass spectrometry
using a VG Quattro Tandem Quadropole mass spectrometer.
XYLA Binds to Calcium
1, a stoichiometry
(n) of 1, and an enthalpy of binding (
H°) of
4.615 kJ mol
1.
Fig. 1.
View of the calcium binding loop of
XYLA. The residues that are the focus of this paper are
represented in ball and stick configurations and are
appropriately labeled. The Ca2+ is in yellow,
and dotted lines depict the electrostatic interactions between the residues within the calcium binding loop of XYLA and the
water molecule.
[View Larger Version of this Image (39K GIF file)]
Fig. 2.
Qualitative analysis of the binding of
calcium to native XYLA and its derivatives. In panels A
and B purified calmodulin (Sigma) (lane 1),
XYLA (lane 2), N261A (lane 3), D262A
(lane 4), D256A (lane 5), and XYLA (lane
6) were subjected to nondenaturing PAGE using a 7.5% (w/v)
polyacrylamide gel. The gels in panels A, C, and
D were stained with Coomassie Blue, and the proteins in
panel B were electroblotted onto a nitrocellulose membrane and then probed with 45Ca as described under
"Experimental Procedures." In panel C, XYLA (lane
1) and XYLA
(lane 2) were pretreated with 10 mM EDTA2 (a) or 10 mM
CaCl2 (b)
and then subjected to nondenaturing PAGE using a 7.5% (w/v) gel. In panel D, purified XYLA
(lane 1), D256A (lane 2), N261A (lane
3), D262A (lane 4), XYLA
(lane 5), and
XYLA/Cex (lane 6) were subjected to SDS-PAGE using a 10% (w/v) polyacrylamide gel. Sigma low molecular weight markers were run
in lane M. Approximately 4 or 10 µg of purified protein
was loaded into each lane of the polyacrylamide gels used in SDS- or
native PAGE, respectively.
[View Larger Version of this Image (53K GIF file)]
Fig. 3.
ITC analysis of binding of calcium to XYLA.
Panel A shows the calorimetric titrations of XYLA (135 µM) with 25 × 10-µl injections of 8 mM CaCl2 in Buffer A (50 mM
Tris/HCl buffer, pH 7.5, containing 100 mM NaCl) at
25 °C (1); 13 × 10-µl injections of Buffer A into
protein solution (protein dilution) (2); 13 × 10-µl
injections of 8 mM CaCl2 into Buffer A (calcium
dilution) (3). Panel B displays the integrated
injection heats from panel A (1), corrected for
control dilution heats (panel A (2) and
(3)). The solid line is the best fit curve that
was used to derive parameters n, Ka, and
H°.
[View Larger Version of this Image (19K GIF file)]
1
and O
2), Asn-261 (O
1), Asp-262 (O
1), and the carbonyl oxygens
of Asn-253 and Asn-258 coordinate with the Ca2+ ion (Fig.
1). The domain does not appear to exhibit a classical EF-hand motif,
which is characterized by three or four glutamate or aspartate residues
coordinating with calcium within a helix-loop-helix structure. To
establish the importance of Asp-256, Asn-261, and Asp-262 in ligand
binding, site-directed mutagenesis was used to construct three variants
of XYLA containing D256A, N261A, and D262A mutations, respectively. A
fourth mutant, designated XYLA
, in which all three residues were
replaced by alanine, was also generated. The different migration
patterns of the native and mutant forms of the xylanase, observed when
the enzymes were subjected to nondenaturing gel electrophoresis (Fig.
2A), could reflect differences in either the overall charge
and/or the size of the XYLA derivatives. Both ITC and 45Ca
autoradiography of XYLA variants blotted onto nitrocellulose membranes
(Fig. 2B) showed that XYLA
and each of the mutants containing single amino acid substitutions did not bind calcium. These
data suggest that all three amino acids play an important role in
calcium binding and support the view that the calcium binding domain of
XYLA is located in the extended loop prior to
-helix 7.
were purified to apparent
homogeneity (Fig. 2D), and the activity of the two enzymes
in the presence of calcium or EDTA was determined. The data (Table
I) showed that the catalytic properties of the two forms
of XYLA were very similar and were not influenced by either EDTA or
calcium. The products released from xylohexaose, by XYLA (in the
presence of 1 mM Ca2+ or 1 mM EDTA)
and XYLA
, over time, were indistinguishable (data not shown). These
data indicate that the calcium binding domain of XYLA does not play an
important role in the catalytic activity of the enzyme.
Enzyme
Ca2+/EDTA
PNPCa
Xylan
Km
kcat
Km
kcat
mM
min
1
mg/ml
min
1
XYLA
1 mM
Ca2+
40
50
0.7
21,000
XYLA
1 mM
EDTA
40
63
1.0
22,000
XYLA
1 mM
Ca2+
50
100
1.1
28,000
XYLA
1
mM EDTA
50
100
1.4
34,000
XYLA/Cex
1
mM Ca2+
40
63
1.1
17,000
XYLA/Cex
1 mM
EDTA
40
63
0.9
17,000
a
PNPC, 4-nitrophenyl- -cellobioside.
To investigate whether the calcium binding
domain played an important role in the thermostability of the enzyme,
the activity of native and mutant forms of XYLA was determined at
different temperatures. In the presence of calcium, XYLA exhibited
increased thermostability compared with the enzyme incubated with EDTA
(Fig. 4). The first order rate constant
(Kinact) for thermal inactivation of XYLA in the
presence of 1 mM EDTA and 1 mM calcium at
57 °C was 5.1 s1 and 0.24 s
1,
respectively (Table II). The protection afforded by
calcium exhibited saturation kinetics (Fig. 5) with the
divalent ion having its maximal effect at a concentration of
approximately 1 mM and above. These data clearly show that
the association of the calcium binding domain with its ligand
stabilizes the enzyme against thermal inactivation.
|
To establish whether the substitution of the key residues in the
calcium binding domain of XYLA influenced the rate of thermal inactivation, the Kinact of D256A, N261A, D262A,
and XYLA at 57 °C was determined. The data, presented in Table
II, showed that although XYLA
, in the presence and absence of
calcium, had Kinact values similar to those of
to Ca2+-XYLA, the other three variants were more prone to
thermal inactivation, both in the presence and absence of calcium.
These data indicate that Asp-256, Asn-261, and Asp-262 interact with
each other to destabilize loop 7, resulting in an increase in the
enzyme's susceptibility to thermal inactivation. These deleterious
interactions can be prevented by either the binding of calcium to loop
7 or the replacement of all three amino acids by alanine.
Although calcium stabilizes XYLA from thermal inactivation, it is not
clear whether this involves a local effect that influences the
structure of the active site or whether the divalent ion stabilizes the
complete structure of the enzyme. To investigate the effect of calcium
on the tertiary structure of XYLA, CD spectroscopy and DSC were
employed. CD spectra of XYLA, treated for 30 min at 57 °C in the
absence of calcium, indicated that the enzyme had very little secondary
structure, suggesting that the protein was essentially unfolded (Fig.
6). In contrast, the CD spectra of XYLA-Ca2+
and XYLA (±CaCl2), with and without pretreatment for 30 min at 57 °C, were very similar, suggesting that the higher
temperature did not cause a significant unfolding of the two proteins.
The stability of XYLA and its derivatives toward thermal unfolding was
also measured using DSC. The thermal denaturation temperature (Tm) of these proteins are shown in Table
III and Fig. 7. The data showed that
removal of calcium from XYLA resulted in an 6 °C drop in the
Tm (Fig. 7A). In the absence and presence
of the divalent cation XYLA
had a Tm similar to
that of XYLA-Ca2+ (Fig. 7B). The
Tm values of D256A, N261A, and D262A were
significantly lower than XYLA-Ca2+ or XYLA
and were
unaffected by the presence or absence of calcium. These data indicate
that the binding of calcium to XYLA causes a gross stabilization of the
protein which results in a decrease in the thermal inactivation of the
enzyme. Similarly, replacing the three amino acids that bind to the
metal ion with alanine stabilizes the three-dimensional structure of
the xylanase against thermal unfolding in the absence of calcium.
|
Proteolysis of XYLA
To establish whether calcium stabilizes
the enzyme against proteinases, the susceptibility of XYLA to
proteolytic inactivation in the presence of excess calcium or EDTA was
assessed. The data, presented in Fig. 8, showed that the
divalent ion protected the xylanase from chymotrypsin attack. The
protection afforded by calcium exhibited saturation kinetics (Fig. 8);
at concentrations 1 mM Ca2+ XYLA was
completely resistant to proteolytic attack. Disruption of the calcium
binding domain by replacing Asp-256, Asn-261, Asp-262, or all three
residues with alanine increased the enzyme's susceptibility to
proteinase inactivation in the presence of calcium (Table II). To
investigate whether chymotrypsin cleaved a specific peptide bond within
the calcium binding domain, XYLA was incubated with chymotrypsin in the
presence and absence of Ca2+ and subjected to SDS-PAGE. The
data, presented in Fig. 9, show that the major transient
proteolysis product had a molecular mass of approximately 29 kDa
(29,918 Da as determined by electrospray ionization mass spectrometry)
and an NH2-terminal sequence that matched XYLA. It would
appear, therefore, that cleavage of XYLA within the calcium binding
loop between residues Asp-262 and Tyr-263 (would generate an
NH2-terminal peptide of 29,910 Da) gave rise to the 29-kDa
polypeptide.
Does a Loop Swap between XYLA and Cex Stabilize the Xylanase?
Replacement of loop 7, between residues 252 and 273, with the corresponding smaller loop from the Cellulomonas fimi xylanase, Cex (28), generated a truncated enzyme, designated XYLA/Cex, which had a Mr of 37,000. XYLA/Cex was purified to homogeneity (Fig. 2D), and the biochemical and biophysical properties of the enzyme were investigated. The data, presented in Table I, showed that XYLA/Cex exhibited catalytic properties that were virtually indistinguishable from XYLA. In contrast, XYLA/Cex was more sensitive to both thermal and proteinase inactivation (Table II) than native XYLA bound to calcium.
The data presented in this paper showed that the
Ka of XYLA for calcium is 4.9 × 104 M1, which represents a
relatively weak affinity compared with EF-hand calcium binding domains
that bind their ligands with Ka values of
105-109 M
1 (29). The
low affinity of XYLA for the divalent ion could reflect the environment
of P. fluorescens subsp. cellulosa. The bacterium was isolated from soil samples of neutral pH in which Ca2+
would be at a concentration >1 mM, sufficient to saturate
XYLA (30). Inspection of the location of the calcium binding domain in
the xylanase suggested that its structure may affect the catalytic properties of the enzyme as Tyr-255, which apparently forms part of
subsite B, is positioned on the Ca2+ binding loop (11).
However, the activity of the enzyme was not significantly influenced by
either the disruption of the metal binding domain, through amino acid
substitution or EDTA treatment, or saturation of the domain with its
appropriate ligand. This suggests that either Tyr-255 does not play a
pivotal role in xylose binding at site B, or the change in conformation
of loop 7 through calcium binding does not influence the ability of the
tyrosine residue to participate in substrate binding. It is also
apparent that the conformational changes associated with calcium
binding to loop 7, which influenced the biophysical properties of the enzyme, did not affect the structure of the extended substrate binding
cleft.
The binding of the calcium binding loop to its ligand caused a
substantial effect on the biophysical properties of XYLA. The enzyme
was less sensitive to thermal inactivation, which reflected a general
increase in the stability of the protein, as evidenced by a
considerable increase in the Tm of XYLA in the
presence of the divalent cation. These data are in agreement with
several previous studies demonstrating that calcium stabilizes the
structure of many proteins (9, 10). Substituting Asp-256 or Asn-261 for
alanine resulted in a substantial destabilization of the enzyme, whereas the D262A mutation resulted in a modest reduction in the thermal stability of the enzyme. It could be argued that these data
suggest that Asp-262 is the key residue involved in calcium binding;
however, ligand binding assays clearly showed that replacing Asp-256,
Asn-261, or Asp-262 with alanine resulted in no detectable affinity of
the XYLA variants for the divalent cation. Interestingly, XYLA, in
which Asp-256, Asn-261, and Asp-262 had all been replaced with alanine,
exhibited thermal stability similar to that of native XYLA bound to
calcium. This could reflect the location of Asp-256, Asn-261, and
Asp-262 in loop 7. The electronegative carbonyl group of Asn-261 and
the negatively charged carboxylic groups of Asp-256 and Asp-262 would
repulse each other, resulting in a destabilization of the loop. Either
the binding of calcium to these amino acids or their replacement with
alanine would prevent the repulsive effects from occurring, resulting
in a stabilization of the loop and thus the whole protein. This view is
consistent with the acid-pair hypothesis proposed by Reid and Hodges
(31) which states that the Ca2+ affinity of EF-hand domains
reflects an electrostatic compromise in which the attraction of
divalent cations for anionic ligands, such as carboxylate groups, is
counterbalanced by the interligand repulsion between coordinating
oxygens. As D262A was more stable than D256A and N261A, it is likely
that the major repulsive effects occur between Asp-262 and either
Asp-256 or Asn-261.
Data presented in this report demonstrate that the initial target for
proteinase attack of XYLA is within the calcium binding domain in loop
7. It is likely, therefore, that the binding of calcium to this region
tightens the conformation of the loop, making it less susceptible to
enzymic cleavage. The transient nature of the 29-kDa peptide released
by chymotrypsin suggests that hydrolysis of peptide bonds in the
calcium binding loop caused a significant destabilization of the
structure of XYLA, resulting in the very rapid subsequent hydrolysis of
the molecule by the proteinase. In contrast to XYLA, XYLA was
susceptible to proteinase attack in the presence and absence of
calcium. These data suggest that the calcium binding loop in XYLA
is
more flexible than in the native enzyme (when complexed with its
ligand), making it more accessible to proteinase cleavage.
From the discussion above, it is apparent that the major role of the calcium binding domain in XYLA is to stabilize the extended structure of loop 7, either against thermal unfolding or proteinase attack. As Ca2+ only protects XYLA from thermal inactivation at temperatures above 55 °C, and the natural habitat of Pseudomonas is normally at a temperature lower than 30 °C, it is unlikely that the enhanced thermal stability afforded by calcium plays an important role in the survival of XYLA. We propose that the major function of the calcium binding domain of the xylanase is to protect the enzyme from proteinase attack. This view is supported by the unusual stability of other xylanases, from both mesophilic and thermophilic microorganisms, to proteolytic attack (7), suggesting that this property exerts a strong selection pressure on the evolution of xylanases.
To date only 5 of the 28 known Family 10 xylanases contain an extended
loop 7. Although inspection of the extended loop in XYLA,
Prevotella ruminicola XYLA (XYNA PRERU), Bacteroides
ovatus XYLA (XYNA BACOV), and Bacillus
stearothermophilus XYLN1 and XYN2 (XYN1 BACST and XYN2 BACST,
respectively) did not reveal motifs, such as the EF-hand, which are
characteristic of calcium binding domains, it is apparent that loop 7 in XYLA did bind to the divalent metal ion. As XYN1 and 2 BACST
function at elevated temperatures, and XYNA PRERU and XYNA BACOV in
environments that contain high levels of proteinase activity, it is
likely that loop 7 in these xylanases is stabilized, possibly though
interactions with metal ions or other sequences within the respective
proteins. The central unanswered questions are why do only 5 Family 10 xylanases contain an extended loop 7, and how did the calcium binding
domain in XYLA evolve? Loop 7 may confer specific catalytic properties
on the enzyme. However, the observation that the biochemical
characteristics of XYLA are not altered when the extended loop is
replaced with the corresponding shorter loop from Cex argues against
this view. It is possible that the ancestral protein that gave rise to
Family 10 xylanases contained an extended loop 7 that destabilized the protein, and through natural selection deletions within the loop have
resulted in the evolution of stable xylanases. Mutations in loop 7 of
the ancestral sequence, which gave rise to XYLA, generated a calcium
binding domain that stabilized the loop, hence there was no requirement
for the loop to be reduced in size in the Pseudomonas
enzyme. An alternative possibility is that the extended loop 7, in 5 of
the 28 Family 10 xylanases, is the result of a DNA insertion into a DNA
sequence that gave rise to the genes encoding the 5 enzymes. In
xynA the inserted DNA subsequently acquired mutations such
that it encoded a calcium binding domain in XYLA. To explore this
hypothesis further, the primary structure of Family 10 xylanases were
subject to phylogenetic analysis using parsimony methods. Initially,
all sequence positions (410) were included in the analysis, which
suggested a relationship between the xylanases containing the extended
loop 7 (data not shown). To remove any bias due to the inclusion of
extended loop 7 in the alignments, the corresponding sequence positions
were removed and the analysis repeated (Fig. 10). It is
interesting that the relationship between 4 of the 5 xylanases is
maintained, indicating that the respective genes have evolved from a
common ancestral sequence that contained a DNA insertion in the region
encoding loop 7. However, this conclusion must be viewed with some
caution as it is apparent that there has been considerable horizontal gene transfer between Family 10 xylanase genes (evidenced by the fact
that very similar sequences occur in taxonomically diverse groups of
organisms), and the bootstrap scores suggest that some clades are not
particularly stable. Based on the relationship between the
Pseudomonas xylanase (XYNA PSEFL) and the clade containing the actinomycete (GUX CELFI and XYNA STRLI) and fungal (XYNA PENCH, GUNF FUSOX, and XYNA PENCH) xylanases, one might hypothesize that the
latter had evolved, following loss of the extended loop 7, from XYNA
PSEFL. However, additional sequence information from this region of the
tree is needed to validate this hypothesis.
To conclude, data presented in this report clearly showed that the extended loop 7 of Pseudomonas XYLA was stabilized by a calcium binding loop. Whether the corresponding extended loops of XYNA PRERU, XYNA BACOV, XYN1 BACST, and XYN2 BACST are also stabilized by binding to divalent metal ions, remains to be elucidated.
We thank Dr. Ian Fleet (UMIST) for doing the mass spectrometry and Judy Laurie for doing the NH2-terminal sequencing.