Courtin, Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre LFoRCe, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20 - PO Box 2463,
Trang 1binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases
Sven Cuyvers, Emmie Dornez, Mohammad N Rezaei, Annick Pollet, Jan A Delcour and
Christophe M Courtin
Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Belgium
Introduction
Glycoside hydrolases can possess noncatalytic
polysac-charide binding sites that facilitate attack on the
natu-ral substrate Most of these sites belong to separate
domains, referred to as carbohydrate-binding modules
(CBMs), linked to the catalytic domain through
flexi-ble linker regions Elaborate research has clarified the
functional relevance of these CBMs [1–4] CBMs are
considered to target the enzyme towards specific cell
wall regions and to keep it in proximity of the
sub-strate In some cases, distortion of the substrate
struc-ture by the CBMs is considered to facilitate hydrolysis
[5] CBMs can also be involved in binding the bacterial
cell wall, thereby anchoring the attached enzyme onto the bacterial surface [6,7]
Despite the clear advantage of having CBMs, some glycoside hydrolases consist of a catalytic domain only [8] Studies investigating the structure of carbohydrate-active enzymes have revealed the presence of other substrate binding regions situated on the surface of the structural unit that contains the catalytic site, rather than on an auxiliary domain [9] These substrate bind-ing sites are located at a certain distance from the active site and are called secondary binding sites (SBS) The presence of one or more SBS has been
Keywords
arabinoxylan; GH11; noncatalytic binding;
single domain xylanase; surface binding
Correspondence
C Courtin, Laboratory of Food Chemistry
and Biochemistry & Leuven Food Science
and Nutrition Research Centre (LFoRCe),
Katholieke Universiteit Leuven, Kasteelpark
Arenberg 20 - PO Box 2463, B-3001
Leuven, Belgium
Fax: + 32 16 321997
Tel: +32 16 321917
E-mail: christophe.courtin@biw.kuleuven.be
(Received 12 October 2010, revised 11
January 2011, accepted 20 January 2011)
doi:10.1111/j.1742-4658.2011.08023.x
The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergil-lus nigerglycoside hydrolase family 11 xylanases was studied by site-direc-ted mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates Modification of the SBS resulted
in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates Weakening of the SBS increased Kd values by up to 70-fold in binding affinity tests using natural substrates The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such struc-tural elements can increase the efficiency of these single domain enzymes
on their natural substrates
Abbreviations
AX, arabinoxylan; AzU, unit of enzyme activity on Azo-wheat AX; CBM, carbohydrate-binding module; GH, glycoside hydrolase family; OSX, insoluble xylan from oat spelts; SBS, secondary binding site; X 6, xylohexaose; X 6 U, unit of enzyme activity on xylohexaose;
XAN, Aspergillus niger xylanase; XBS, Bacillus subtilis xylanase; XyU, unit of enzyme activity on Xylazyme AX.
Trang 2reported in enzymes belonging to glycoside hydrolase
family (GH) 8, 10, 11, 13, 14, 15, 16 and 77 [9–20]
The widespread occurrence of these binding sites
indi-cates that incorporation of a SBS provides an
evolu-tionary benefit for these enzymes However, the
function of these SBS in many enzymes remains to be
unraveled To date, most work aiming to understand
the role of SBS has been performed on starch
degrad-ing enzymes Human salivary a-amylase contains
sev-eral SBS Mutational analysis demonstrated that these
SBS residues are important for the activity on starch
and that they play a role in the binding of the enzyme
to bacteria of the oral cavity [15] Nielsen et al [9]
concluded that the two SBS in barley a-amylase each
have a distinct binding specificity, although they both
play a role in substrate targeting In two single domain
glucoamylases from Saccharomycopsis fibuligera, a SBS
was also found to enhance binding to starch granules
[18]
In xylanases (EC 3.2.1.8), the existence of a SBS was
also discovered in several single domain enzymes: one
belonging to GH8 [10], one to GH10 [11] and three to GH11 [12,13] In GH11, the existence of these SBS was recently identified by NMR-monitored titrations
of Bacillus circulans xylanase [13] and X-ray analysis of crystals of catalytically incompetent mutants of the xylanases of Bacillus subtilis (XBS) and Aspergillus niger (XAN) soaked with xylo-oligosaccharides [12] GH11 xylanases have a b-jelly roll fold structure, which is often compared to a partially closed right hand [21] The SBS are present in different regions of the GH11 xylanases In the B circulans xylanase and
in XBS, the SBS is located on the ‘knuckles’ of the enzyme, whereas, in XAN, it is located at the ‘tip of the fingers’ (Fig 1) Because SBS in these enzymes are located distant from the active site, their impact on substrate hydrolysis is expected to be limited to longer substrates
In the present study, the impact of the presence of a SBS on the biochemical properties of single domain GH11 xylanases was investigated by extensive muta-tional analysis of XBS and XAN This allowed the
A
B
Fig 1 Superposition of the overall structures of the xylanases from A niger (green, PDB 2QZ2) and B subtilis (blue, PDB 2QZ3) in complex with oligosaccharides shows the presence of a secondary substrate binding site in different surface regions of these enzymes (on the left) The figure was drawn using PYMOL (http://pymol.sourceforge.net/) On the right, schematic representations are shown of the oligosaccha-rides bound at the secondary binding sites of the A niger xylanase (A) and the B subtilis xylanase (B) The diagrams of protein–ligand inter-actions were generated using LIGPLOT [37] based on Vandermarliere et al [12] Amino acid residues that form direct protein–ligand interactions with their main chain only are indicated by an asterisk.
Trang 3investigation of whether the SBS has a similar
func-tionality in different GH11 xylanases and hence
whether the occurrence of SBS is a more general
strat-egy of GH11 xylanases to compensate for their lack of
CBMs
Results and Discussion
Genetic engineering of the SBS
Residues of the SBS of both XBS and XAN involved
in substrate interaction were selected based on the
crystal structures of XBS and XAN soaked with
xylo-tetraose and xylopentaose, respectively [12] (Fig 1),
and were subjected to genetic engineering using
site-directed mutagenesis Amino acid residues reported to
potentially play a role in secondary substrate binding
were mutated to Ala aiming to investigate their
impor-tance Several mutations were also combined to assess
the importance of the SBS as a whole for the
biochem-ical properties of XBS and XAN In an attempt to
increase substrate binding affinity of the SBS, aromatic
residues were introduced at certain places to create
extra or stronger hydrophobic stacking interactions or
residues were replaced to create new hydrogen bonds
Screening procedure
To examine the impact of different mutations on the
functionality of the SBS in XBS and XAN, a screening
method on nonpurified enzyme samples was developed
Because the SBS is located far from the active site, it
was hypothesized that the hydrolysis of soluble,
oligo-meric substrates, such as xylohexaose (X6), is not
influ-enced by the presence of a SBS because this substrate
cannot interact with both the SBS and the active site
at the same time By contrast, larger polymeric
sub-strates, such as Xylazyme arabinoxylan (AX), can
reach both sites simultaneously Previously, it was
demonstrated that X6binds independently to the active
site and the SBS of the B circulans xylanase, whereas
larger substrates, such as xylododecaose, bind the two
sites cooperatively [13] Accordingly, a screening ratio
was defined as the activity on Xylazyme AX divided
by the activity on X6 This ratio is considered to reflect
the impact of a modification in the SBS on its
func-tionality, independent of the expression efficiency of
the protein Because Escherichia coli and Pichia
pasto-ris do not produce xylanolytic enzymes, this ratio of
two activities enables a comparison of nonpurified
enzymes However, the possibility that other proteins
present in the nonpurified enzyme samples might have
an unforeseen effect on the activity of XBS or XAN
could not entirely be ruled out at this stage The screening procedure was performed on E coli cell lysates containing (mutant) XBS and on P pastoris expression media containing (mutant) XAN The results of the screening for XBS and XAN with a modified SBS are shown in Table 1
XBS The results of the screening clearly show that modifica-tion of the SBS of XBS leads to a lower relative effi-ciency towards the water-unextractable Xylazyme AX compared to that towards X6 These results are in agreement with the observations of Ludwiczek et al [13] on GH11 B circulans xylanase, which has a SBS equivalent to that of XBS Replacement of residues considered to play a role in secondary binding with Ala leads to a lower screening ratio for all enzymes For the G56A-T183A-W185A mutant, a large drop in the screening ratio is seen, resulting in a ratio that is only half the ratio obtained for the wild-type XBS The results shown in Table 1 also demonstrate the importance of the hydrophobic stacking interaction that Trp185 makes with bound substrate Hydrogen bonds with other residues also appear to be of major importance Mutation of residues Thr183, Asn181 and Gly56 to Ala leads to a large drop in the screening ratio A smaller effect is also observed upon mutation
of Asn54 and Asn141 These results correspond well with the results obtained in the previous study by Van-dermarliere et al [12] that reported residues important for secondary substrate binding The results obtained for mutants where an attempt was made to increase substrate binding affinity show that the efficiency on polymeric versus oligomeric substrate was not increased Lower screening ratios emerged for all these mutants compared to the wild-type This shows that the intended fortification of the SBS failed or that a stronger SBS does not lead to more efficient hydrolysis
of polymeric substrate
XAN Screening of XAN mutants where amino acid residues involved in secondary substrate binding are replaced
by Ala shows a trend similar to that observed for the set of XBS mutants, although the differences are smal-ler This indicates that the SBS in both enzymes proba-bly has the same functionality The screening ratio goes down by a maximum of approximately 30% in mutants E31A and Y29A-E31A, whereas, for XBS, decreases of up to 50% were observed Glu31 appears
to be indispensable for the SBS of XAN because the
Trang 4screening ratio obtained for mutant E31A is not
low-ered further when extra Ala mutations are introduced
Glu31 can make several hydrogen bonds with substrate
bound in the SBS [12] (Fig 1) Tyr29, which can make
a hydrophobic stacking interaction with bound
sub-strate, also appears to be an important residue for the
SBS The screening ratio of D32A is similar to that of
the wild-type, indicating the minor importance of the acidic side chain of Asp32 This result is logical because Asp32 makes only one hydrogen bond with surface bound substrate through a main chain amine group that is not abolished by the D32A mutation (Fig 1) The acidic side chain is 3.7 A˚ away from a hydroxyl group of the bound substrate and this dis-tance is too far to form a relevant hydrogen bond [12] The introduction of amino acid residues to create new
or stronger hydrophobic stacking of hydrogen bonds with substrate in the SBS has led to screening ratios similar to that of the wild-type XAN for most enzymes Subtle changes in the hydrogen bonding appear to have no (or only a very minor) effect on the functionality of the SBS In some cases, the introduc-tion of aromatic residues even lowered the screening ratio, as was seen for some of the XBS mutants
Activity measurements After the screening procedure, a smaller set of enzymes was selected for purification and further biochemical characterization The activity of these enzymes was determined on X6 and two chromophoric polymeric substrates: the water-unextractable Xylazyme AX and the water-extractable Azo-wheat AX Table 2 lists these results along with temperature and pH optima of the enzymes Most mutations lead to a lower tempera-ture optimum, whereas little or no change is observed
in the pH optimum The lowered temperature opti-mum is possibly explained by decreased enzyme sta-bility at higher temperatures The solubilization of water-unextractable AX isolated from wheat flour and insoluble oat spelt xylan (OSX) by the different mutants was also examined For several mutants, the solubilization in function of the enzyme concentration used in the assay is shown in Fig 2
XBS Mutations in the SBS of XBS appear to have no (or very little) effect on the activity on X6because all XBS mutants give results similar to those of the wild-type xylanase The substrate is probably too small, so the enzyme cannot benefit from the presence of an addi-tional SBS at the enzyme surface However, the activi-ties on Xylazyme AX and Azo-wheat AX drop upon modification of the SBS These results suggest that the functionality of the SBS is limited to larger substrates that can reach both the active site and SBS at the same time Because the activity on X6 remains the same upon engineering of the SBS of XBS, the results obtained in the previous screening experiment directly
Table 1 The effect of genetic engineering of the secondary
bind-ing site of the B subtilis and A niger xylanases on the screenbind-ing
ratio The screening ratio is defined as the ratio of activity on
Xyla-zyme AX and activity on X6 Screening ratios are expressed relative
to the ratio of the wild-type enzyme (100%) and were calculated
based on two independent activity measurements on X6and two
independent activity measurements on Xylazyme AX on the same
unpurified enzyme sample, with each independent assay
compris-ing three replicates Data are shown as the mean ± SD.
Screening ratio (%) XBS
XAN
*Significantly different from the wild-type enzyme by Student’s
t-test (P < 0.05) To account for multiple comparisons, the
signifi-cance levels were adjusted according to Scheffe´’s method.
Trang 5reflect the activity of the enzymes on Xylazyme AX.
The activity drop on polymeric substrates is the largest
for the G56A-T183A-W185A, mutant with an activity
that dropped to half on Xylazyme AX and even to
one-third on Azo-wheat AX compared to that of
wild-type XBS The trends observed on chromophoric
substrates are confirmed on natural substrates because
modification of the SBS decreases the rate at which
XBS solubilizes water-unextractable AX and OSX
Especially in the case of OSX, the solubilization by the
enzymes is greatly hampered upon modification of the
SBS At the same enzyme concentration, the wild-type
XBS solubilized the most The maximal attainable
sol-ubilization of these substrates by different mutants was
also measured, although no clear differences were
observed (results not shown) This indicates that the
SBS influences the rate of hydrolysis, most likely by
enhancing substrate recognition, rather than affecting
the real catalytic potential and substrate specificity of
the enzyme
XAN
By contrast to results on XBS, the activity on X6 is affected by a number of the mutations made in the SBS of XAN A much lower activity is observed espe-cially for those enzymes containing the E31A mutation display This single mutation leads to an activity on X6
that is only half that of the wild-type XAN However,
a loss of activity on X6 in the set of purified XAN mutants does not appear to be correlated with a weak-ening of the SBS For example, Y29A does not display
a lower activity on X6, whereas it is regarded as one of the most important residues for secondary binding The decrease also cannot be explained by differences
in enzyme stability under the assay conditions (Fig S1) The small decrease in activity under assay conditions observed for a few XAN mutants is not proportional to the decrease in activity on X6 The loss
of activity on X6 for certain mutants might be attrib-uted to these mutations exerting an effect on the
Table 2 Biochemical characterization of B subtilis and A niger xylanases with a modified secondary binding site Values shown are expressed relative to the activity of the wild-type enzyme (100%) The activity on X 6 was calculated from values obtained from three inde-pendent trials, each comprising five indeinde-pendent samples; the activity on Xylazyme AX and Azo-wheat AX was from five indeinde-pendent trials, each comprising three replicates For each enzyme, values with the same letter in one column are not significantly different from each other according to Tukey’s tests (P < 0.05) performed with SAS , version 9.2 (SAS Institute) For the wild-type XBS, 1 X 6 U = 1.34 · 10)10M ,
1 XyU = 9.76 · 10)10M and 1 AzU = 50.8 · 10)10M and for the wild-type XAN, 1 X6U = 1.02 · 10)10M , 1 XyU = 7.72 · 10)10M and
1 AzU = 9.65 · 10)10M for the activity on X6, Xylazyme AX and Azo-wheat AX, respectively (data are shown as the mean ± SD) Kdvalues are expressed in mgÆmL)1and are apparent K d values in many cases as a result of substrate concentration limitations in the test (data are shown as the mean ± SE from the fit on a single curve) The reported temperature and pH ranges indicate the intervals in which the observed activity was at least 95% of the maximal activity of the enzyme.
Xylohexaose
Xylazyme AX
Azo-wheat AX
Water-unextractable AX OSX Temperature pH XBS
XAN
Trang 6overall catalytic efficiency of the enzyme Changes of
SBS residues located on the outer b-sheet of the XAN
structure might induce subtle changes in the position
of important binding or catalytic residues in the active
site located on the inner b-sheet of the b-jelly roll The
results for the activity on Xylazyme AX and
Azo-wheat AX in Table 2 therefore do not show clear
trends at first sight The ratios of activities on
Xyla-zyme AX over X6, however, do confirm the results of
the screening Solubilization experiments with the
nat-ural substrates OSX and water-unextractable AX also
clearly demonstrate that the solubilizing capacity is
strongly decreased upon modification of SBS residues
The observed drops in solubilizing capacity are
espe-cially spectacular on OSX
Comparison of XBS and XAN
For XBS, it is clear that the residues involved in
sec-ondary substrate binding play no (or a very minor)
role in the hydrolysis of oligomeric substrates The SBS is located too far from the active site to influence the binding and catalysis of these substrates in the active site The same statement is probably true for most residues in the SBS of XAN, although, in this case, some mutants (especially those containing the E31A mutation) display a strong decrease in activity
on X6 The reason for this is not clear Possibly, these mutations provoke subtle positional changes of resi-dues located in the active site The results of activity measurements on purified enzymes, as presented in the present study, support the results already obtained by screening and thereby indicate that the screening pro-cedure can indeed provide a valuable tool for the ini-tial selection of mutant enzymes The SBS in XBS and XAN mainly function to increase the efficiency of the enzyme on polymeric substrates, as demonstrated by the results obtained for both chromophoric and natu-ral substrates The drop in activity on polymeric sub-strates upon mutations in the SBS is substantial,
0 10 20 30 40
0 10 20 30 40
Enzyme concentration ( ×10 –10 M )
Enzyme concentration ( ×10 –8 M )
0 10 20 30 40
0 10 20 30 40
LEGEND
XBS wild-type XBS W185A XBS G56A-T183A-W185A
XBS N54W XBS N54W-N141Q
LEGEND
XAN wild-type XAN Y29A XAN E31A
XAN Y29A-E31A XAN D16A-Y29A-E31A
A
Enzyme concentration ( ×10 –9 M )
0 10 20 30 40
0 10 20 30 40
C
D
Enzyme concentration ( ×10 –8 M )
0 10 20 30 40
0 10 20 30 40
B
Fig 2 Solubilization of water-unextractable arabinoxylan (WU-AX) (A) and oat spelt xylan (OSX) (B) by B subtilis xylanase mutants with a modified secondary binding site and of water-unextractable arabinoxylan (C) and oat spelt xylan (D) by A niger xylanase mutants.
Trang 7especially when considering that these mutations are
located far from the active site In general, the drop in
activity on Azo-wheat AX is slightly larger than that
on Xylazyme AX, as shown in Table 2 One parameter
that is often used to refer to the ratio of activity
towards water-unextractable and water-extractable AX
is substrate selectivity [22] It is often expressed as a
substrate selectivity factor, which can be calculated as
the activity on Xylazyme AX over the activity on
Azo-wheat AX [22], and is a determinant for functionality
of xylanases in several applications [23] Table 3 lists
substrate selectivity factors for XBS and XAN with a
modified SBS Although the differences are small, a
general trend can be seen in which weakening of the
SBS increases substrate selectivity In XBS, especially
for those mutants containing W185A, significant
dif-ferences in substrate selectivity are observed In XAN,
the differences are smaller, although the same general
trend is seen Water-unextractable AX are probably
less flexible and therefore it might be more difficult
for the SBS to exploit its full functionality with respect
to the hydrolysis of these substrates Whether the
observed differences in substrate selectivity are relevant
in applications remains to be explored Strikingly, the
W185A mutation in XBS has already been
character-ized with regard to its effect on substrate selectivity
However, Moers et al [24] reported a drop in the
sub-strate selectivity factor for the W185A mutant One
possible explanation for this discrepancy could be the
use of a His-tagged protein in their study The
C-ter-minal location of this His-tag suggests a likely
interfer-ence with the functionality of the SBS because three
important residues for secondary binding are located
near the C-terminus
Binding affinity towards insoluble polymers
As outlined above, the effect of the SBS on activity
towards different substrates was studied Obviously,
substrate binding is closely linked to activity
There-fore, the binding of the different XBS and XAN
mutants towards water-unextractable AX and OSX
was assessed by constructing binding curves, as shown
for several mutants in Fig 3 Table 2 gives the overall
dissociation constants (Kd) derived from these curves
XBS
Weakening of the SBS of XBS clearly increases Kd
val-ues and therefore lower affinities towards both
water-unextractable AX and OSX The differences on OSX
are more pronounced than those on
water-unextract-able AX The G56A-T183A-W185A mutant has a Kd
of 25 mgÆmL)1 on water-unextractable AX and
29 mgÆmL)1 on OSX, respectively, compared to 8.8 mgÆmL)1 and 0.4 mgÆmL)1 for the wild-type XBS
It can probably be regarded as an enzyme without a SBS The elimination of the possibility of substrate forming a hydrophobic stacking interaction with Trp185 (W185A) leads to the largest increase in Kd for a single mutation, giving rise to Kd values of
17 mgÆmL)1 on water-unextractable AX and
13 mgÆmL)1 on OSX On water-unextractable AX, N54W and N141Q appear to have slightly lower Kd values, similar to the combined N54W-N141Q mutant, which has a Kdof 5.3 mgÆmL)1 By contrast, on OSX, all mutants give higher Kd values than the wild-type enzyme It is difficult to pinpoint the reason for the higher affinity of these mutants towards water-unex-tractable AX One possibility is that the attempt to enhance substrate binding was successful for water-unextractable AX, although a higher affinity is not necessarily correlated with a higher activity
Table 3 Substrate selectivity factors of B subtilis and A niger xylanases with a modified secondary binding site The substrate selectivity factor is calculated as the ratio of activity on Xylazyme
AX over the activity on Azo-wheat AX, with both activities calcu-lated based on the values obtained from five independent trials, each comprising three replicates All values are expressed relative
to the wild-type enzyme (1.00) Data are shown as the mean ± SD.
Substrate selectivity factor XBS
XAN
*Significantly different from the wild-type enzyme by Student’s t-test (P < 0.05) To account for multiple comparisons, the signifi-cance levels were adjusted according to Scheffe´’s method.
Trang 8A substrate may be bound too tightly to the enzyme
to allow efficient hydrolysis For CBMs, it has also
been suggested that too strong a binding affinity
between substrate and CBM may limit the activity of
the attached enzyme [1,25] Another possibility is that
these mutations result in stronger substrate binding
but that this binding, for example, orients the
sub-strate wrongly to assist in its catalysis in the active
site Binding experiments have shown that N54W,
N141Q and N54W-N141Q also display higher affinity
towards some other polysaccharides such as cellulose
and barley b-glucan than the wild-type XBS (results
not shown) This might indicate that these mutations
create a ‘sticky patch’ causing aspecific binding to all
kinds of substrates, rather than enhancing the specific
binding of xylan substrates in a correct orientation to
help provide the catalytic site with substrate for
hydrolysis
XAN Affinity towards both water-unextractable AX and OSX is decreased upon modification of the SBS of XAN The wild-type XAN has lower Kd values than mutants that weaken the SBS, as well as mutants aimed at creating a SBS with increased substrate bind-ing affinity The Kd of wild-type XAN is 24 mgÆmL)1 for water-unextractable AX and 3.8 mgÆmL)1 for OSX The Y29A mutation increases the Kd values to
93 mgÆmL)1 and 41 mgÆmL)1 for water-unextractable
AX and OSX, respectively The largest increase is seen for the D16A-Y29A-E31A mutant, with Kd values of
122 mgÆmL)1 and 61 mgÆmL)1 on water-unextractable
AX and OSX, respectively The binding of mutants aimed at obtaining an enzyme with an increased sub-strate binding affinity in the SBS also appeared to be negatively affected However, for most of these
0 20 40 60 80 100
0 10 20 30 40
WU-AX (mg·mL)
0 20 40 60 80 100
0 10 20 30 40
OSX (mg·mL) LEGEND
Wild-type XAN XAN Y29A XAN E31A
XAN Y29A-E31A XAN D16A-Y29A-E31A
LEGEND
Wild-type XBS XBS W185A XBS G56A-T183A-W185A
XBS N54W XBS N54W-N141Q
A
0 20
40
60
80
100
0 10 20 30 40
OSX (mg·mL)
WU-AX (mg·mL)
0 20 40 60 80 100
0 10 20 30 40
C
Fig 3 Binding of B subtilis xylanase mutants with a modified secondary binding site to water-unextractable arabinoxylan (WU-AX) (A) and oat spelt xylan (OSX) (B) and of A niger xylanase mutants to water-unextractable arabinoxylan (C) and oat spelt xylan (D).
Trang 9enzymes, this is not reflected by the activity
measure-ments Unexpectedly, Kd values of D32A are higher
than those of the wild-type, whereas the activity of the
mutant was unaffected
Comparison of XBS and XAN
In general, large differences in affinity are observed for
both XBS and XAN upon modification of the SBS
Even single mutations in the SBS can lead to a drastic
increase in the Kdvalue Aromatic residues (Trp185 in
XBS and Tyr29 in XAN) appear to play an essential role
in the substrate binding In most cases, a lower activity
and a lower affinity appear to be satisfactorily
corre-lated However, in XBS, stronger binding does not
nec-essarily increase activity This result suggests that the
function of the SBS is not merely to bring the enzyme
into contact with insoluble substrates, as is often
pro-posed as one of the functions of CBMs [1,2] The SBS
possibly has a more pronounced role, such as assisting
catalysis by leading the substrate into the active site
Relevance of the present findings
Many glycoside hydrolases contain one or more CBMs
that are considered to function as an aid to target
sub-strates and to keep enzymes in proximity with their
substrate [1,2] The discovery of a SBS in two single
domain xylanases from B subtilis and A niger led to
the suggestion that these structures compensate for the
lack of CBMs in these enzymes [12] In the B circulans
xylanase, it was found that the SBS assists the active
site by binding larger substrates cooperatively, thereby
facilitating their hydrolysis [13] In the present study,
the demonstrated effects of modification in the SBS of
XBS and XAN on binding affinities, as well as on
activity, indicate that these sites are of significant
importance for the enzymes Previously, the deletion of
CBMs from (or fusion of CBMs to) xylanases was
shown to lead to lower activities in the absence of the
CBM comparable to those seen for the elimination of
the SBS in the present study [26–28] In most studies,
however, the presence of CBMs is correlated with a
higher activity on insoluble substrate, whereas the
activity on soluble substrate is often unaffected [26,27]
However, in the present study, the presence of a SBS
gives rise to higher activities on all tested polymeric
substrates (i.e both extractable and
water-unextractable) The presence of a SBS was even more
beneficial for enzyme activity on water-extractable
sub-strate This difference might be explained by the fact
that a water-extractable AX chain is probably more
flexible and therefore can be guided more easily into
the active site cleft once it is bound to the SBS Although the exact functional role of the SBS in GH11 xylanases is not clear from the data obtained in the present study, it might be speculated that SBS is involved in targeting the enzymes towards their sub-strate and, subsequently, in anchoring them onto the substrate or in feeding the substrate chain to the active site cleft, corresponding to their suggested role in bar-ley a-amylase [29] Whatever the case, the demon-strated effect of the SBS on affinity and activity towards polymeric substrate confirms that the sites are
of great assistance to XBS and XAN with respect to overcoming the lack of CBMs
The large beneficial effect of a SBS and its presence
in two different regions of two different single domain GH11 xylanases leads to the assumption that XBS and XAN are representatives of a larger group of single domain xylanases that have evolved this feature The sequence alignment of known GH11 enzymes reported
by Sapag et al [30] reveals that residues important for SBS in XBS and XAN also occur in other single domain xylanases The residues important for second-ary substrate binding in XAN occur in a subgroup of fungal GH11 that is defined as ‘group II’ by Sapag
et al [30] The SBS of XBS appears to be present in several xylanases of other Bacillus species Further-more, it is possible that other GH11 xylanases also contain undiscovered SBS in different regions of the enzyme The concept of a SBS in GH11 xylanases demonstrated in the present study is possibly also valid for other single domain xylanases As noted in the Introduction, the presence of a SBS has also been sug-gested in GH8 and GH10 xylanases [10,11] Future work on these enzymes will aim to clarify whether the SBS has the same functional relevance for these enzymes
Conclusions
Screening of a large set of XBS and XAN with a mod-ified SBS clearly established that the SBS raises the rel-ative activity of single domain xylanases on polymeric versus oligomeric substrate Activity measurements on purified enzymes confirmed these findings For XBS, the activity on X6 is independent of the strength of the SBS; for XAN, this is probably also the case, although, for a few mutations, the overall activity was seriously decreased Although the differences are small, the efficiency of the SBS in XBS and XAN appears to
be higher for water-extractable substrate, probably because it is less rigid or more accessible than water-unextractable substrate, which is tightly associated with other components in the cell wall matrix
Trang 10The effects on activity on different substrates and
the binding affinity towards the natural substrates,
water-unextractable AX and OSX, as described in the
present study, imply that the SBS is of significant
importance for XBS and XAN The SBS in these
single domain xylanases may be an example of a very
efficient strategy that targets the enzyme towards a
substrate to anchor and feed it into the active site,
which are functions often attributed to CBMs in
multi-domain enzymes Future work will need to unravel the
mechanism by which this SBS can assist hydrolysis In
addition, the extent to which SBS occur amongst
xy-lanases and other glycoside hydrolases will need to be
determined
Materials and methods
Materials
All chemicals, solvents and reagents were purchased from
Sigma-Aldrich (Bornem, Belgium) and are of analytical
grade, unless specified otherwise Xylazyme AX tablets,
liquid Azo-wheat AX, water-unextractable AX isolated
from wheat flour, barley b-glucan and xylooligosaccharides
up to X6 were obtained from Megazyme (Bray, Ireland)
Pustulan was obtained from Calbiochem (Darmstadt,
Ger-many) Oligonucleotide primers were obtained from
Sigma-Aldrich The insoluble fraction of oat spelt xylan was
obtained by removing the soluble component from the
starting material First, an extract was made of 1.0 mg
xylan per 20 mL of water by shaking the solution for
15 min at 4C After centrifugation (1500 g for 10 min),
the residue was boiled for 30 min in 20 mL water per
milli-gram of starting material After a new centrifugation step
(11000 g for 30 min), the remaining soluble components
were removed from the residue by shaking in 20 mL of
water per milligram of starting material for 15 min at room
temperature After centrifugation (11000 g for 30 min), the
insoluble fraction was lyophilized
Site-directed mutagenesis
Expression plasmid pEXP5-CT-xyna was used for
heterolo-gous expression of XBS (UniProtKB P18429) in E coli
[31] For the heterologous expression of XAN (differing in
three amino acids from UniProtKB P55329, namely K50N,
E57D and M167V) in P pastoris, the pPicZaC-exlA
plas-mid was used [32] In both plasplas-mids, a stop codon was
incorporated after the last nucleotide encoding for the
C-terminal amino acid of the native protein (Trp185 and
Ser184 for XBS and XAN, respectively) All mutants were
constructed using a QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA, USA) The template DNA
and oligonucleotide primers used are shown in Table S1
E coli TOP10 cells (Invitrogen, Groningen, the Nether-lands) were transformed with the modified plasmids (3 lL)
by heat shock (30 s at 42C) Success of mutagenesis was verified by sequence analysis (Genetic Service Facility, VIB, Wilrijk, Belgium)
Recombinant expression XBS
E coli* BL21 (DE3) pLysS cells transformed with pEXP5-CT-xynA (or mutant constructs) were used to express XBS (and its mutant variants) in accordance with a method previously described by Pollet et al [33] The enzyme yield
of recombinant XBS after purification was typically 20–50 mgÆL)1culture
XAN PmeI (New England Biolabs, Beverly, MA, USA) linearized pPicZaC-exlA was used to transform P pastoris strain X33
by electroporation Extracellular expression of XAN was performed using the EasySelect Pichia expression kit (Invi-trogen) in accordance with the manufacturer’s instructions More specifically, 500 lL of an overnight culture of trans-formed cells in ‘yeast extract peptone dextrose medium’ containing 100 lgÆmL)1 Zeocine (InvivoGen Cayla, Tou-louse, France) was used for the inoculation of 90 mL of
‘buffered complex medium containing glycerol’ The result-ing culture was then grown for 16–20 h at 30C under con-tinuous shaking before the cells were harvested by centrifugation (2500 g for 5 min) and transferred to 20 mL
of ‘buffered complex medium containing methanol’ for induction of protein expression This culture was then incu-bated for an additional 4 days at 30C and 400 lL of pure methanol was added each day After centrifugation (2500 g for 10 min), the supernatant was collected for further puri-fication (see below) One culture typically yielded 5–15 mg
of XAN after purification
Protein purification XBS
XBS and its mutant variants were purified from the E coli cell lysates with cation exchange chromatography, as previ-ously described by Pollet et al [33] To remove the last con-taminating proteins, an additional gel filtration step was performed on a Sephacryl S-100 column (GE Healthcare, Uppsala, Sweden) with sodium acetate buffer (250 mm,
pH 5.0) as elution buffer
XAN XAN and its mutant variants were purified with anion exchange chromatography, as previously described by