Secondary substrate binding strongly affects activity and binding 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 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 The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed 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 structural elements can increase the efficiency of these single domain enzymes on their natural substrates (Received 12 October 2010, revised 11 January 2011, accepted 20 January 2011) doi:10.1111/j.1742-4658.2011.08023.x Introduction Glycoside hydrolases can possess noncatalytic polysaccharide binding sites that facilitate attack on the natural substrate Most of these sites belong to separate domains, referred to as carbohydrate-binding modules (CBMs), linked to the catalytic domain through flexible 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 substrate In some cases, distortion of the substrate structure 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 carbohydrateactive 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 binding 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 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; X6, xylohexaose; X6U, unit of enzyme activity on xylohexaose; XAN, Aspergillus niger xylanase; XBS, Bacillus subtilis xylanase; XyU, unit of enzyme activity on Xylazyme AX 1098 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al Secondary substrate binding in GH11 xylanases reported 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 indicates that incorporation of a SBS provides an evolutionary 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 degrading enzymes Human salivary a-amylase contains several 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 mutational analysis of XBS and XAN This allowed the A B Fig 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 oligosaccharides bound at the secondary binding sites of the A niger xylanase (A) and the B subtilis xylanase (B) The diagrams of protein–ligand interactions 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 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1099 Secondary substrate binding in GH11 xylanases S Cuyvers et al investigation of whether the SBS has a similar functionality in different GH11 xylanases and hence whether the occurrence of SBS is a more general strategy of GH11 xylanases to compensate for their lack of CBMs 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 Results and Discussion XBS 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 xylotetraose and xylopentaose, respectively [12] (Fig 1), and were subjected to genetic engineering using sitedirected mutagenesis Amino acid residues reported to potentially play a role in secondary substrate binding were mutated to Ala aiming to investigate their importance Several mutations were also combined to assess the importance of the SBS as a whole for the biochemical 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, oligomeric substrates, such as xylohexaose (X6), is not influenced 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 substrates, such as Xylazyme arabinoxylan (AX), can reach both sites simultaneously Previously, it was demonstrated that X6 binds 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 functionality, independent of the expression efficiency of the protein Because Escherichia coli and Pichia pastoris 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 1100 The results of the screening clearly show that modification of the SBS of XBS leads to a lower relative efficiency 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 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 Vandermarliere 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 smaller This indicates that the SBS in both enzymes probably 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 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al Secondary substrate binding in GH11 xylanases Table The effect of genetic engineering of the secondary binding site of the B subtilis and A niger xylanases on the screening ratio The screening ratio is defined as the ratio of activity on Xylazyme 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 X6 and two independent activity measurements on Xylazyme AX on the same unpurified enzyme sample, with each independent assay comprising three replicates Data are shown as the mean ± SD Screening ratio (%) XBS Wild-type N54A G56A N141A N181A T183A W185A N54A-G56A N54A-T183A N181A-T183A G56A-T183A-W185A N54F N54W N141Q N54W-N141Q XAN Wild-type D16A Y29A E31A D32A D16A-E31A Y29A-E31A D16A-Y29A-E31A G15W D16Y Y29W E31Q E31T D32E D32F D32N D32Q D32W 100 86 70 81 70 63 62 68 65 66 54 83 76 80 79 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13 19 15* 13 21 13* 12* 13* 16* 23 14* 10 17 26 21 100 92 82 70 101 76 73 82 98 83 89 101 104 105 86 99 98 95 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13 13* 16 5* 5* 12 8 10 13 11 11 *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 screening ratio obtained for mutant E31A is not lowered 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 substrate, 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 distance 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 introduction 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 lists these results along with temperature and pH optima of the enzymes Most mutations lead to a lower temperature optimum, whereas little or no change is observed in the pH optimum The lowered temperature optimum is possibly explained by decreased enzyme stability 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 XBS Mutations in the SBS of XBS appear to have no (or very little) effect on the activity on X6 because 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 additional SBS at the enzyme surface However, the activities 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 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1101 Secondary substrate binding in GH11 xylanases S Cuyvers et al Table 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 X6 was calculated from values obtained from three independent trials, each comprising five independent samples; the activity on Xylazyme AX and Azo-wheat AX was from five independent 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, X6U = 1.34 · 10)10 M, XyU = 9.76 · 10)10 M and AzU = 50.8 · 10)10 M and for the wild-type XAN, X6U = 1.02 · 10)10 M, XyU = 7.72 · 10)10 M and AzU = 9.65 · 10)10 M for the activity on X6, Xylazyme AX and Azo-wheat AX, respectively (data are shown as the mean ± SD) Kd values are expressed in mgỈmL)1 and are apparent Kd 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 Activity (%) on Xylazyme AX Xylohexaose XBS Wild-type N181A T183A W185A G56A-T183A-W185A N54W N141Q N54W-N141Q XAN Wild-type D16A Y29A E31A D32A Y29A-E31A D16A-Y29A-E31A Y29W E31Q E31T D32E Affinity (Kd) towards Azo-wheat AX Waterunextractable AX OSX Temperature pH 100 113 100 100 94 102 103 102 ± ± ± ± ± ± ± ± AC C AB AB A B AB B 100 81 73 70 52 89 96 92 ± ± ± ± ± ± ± ± 6 E C B B A D DE DE 100 77 66 53 33 85 92 92 ± ± ± ± ± ± ± ± 8 14 11 E CD C B A DE DE DE 8.8 11 11 17 25 8.1 6.1 5.3 ± ± ± ± ± ± ± ± 0.4 2 4.5 0.5 0.3 0.4 0.4 8.1 6.2 13 29 5.6 1.9 2.5 ± ± ± ± ± ± ± ± 0.01 0.6 0.3 0.4 0.1 0.2 48–54 43–49 38–42 39–46 37–42 43–50 47–52 46–51 °C °C °C °C °C °C °C °C 5.2–6.8 5.2–6.8 5.2–6.6 5.3–6.6 5.2–6.5 5.3–6.6 5.2–6.8 5.2–6.8 100 86 97 51 102 61 39 111 94 102 77 ± ± ± ± ± ± ± ± ± ± ± 10 19 11 12 10 DE DE DE B E C A E DE E D 100 85 80 44 99 50 35 102 99 103 72 ± ± ± ± ± ± ± ± ± ± ± 5 2 4 F E E B F C A F F F D 100 82 72 45 91 48 29 98 93 99 77 ± ± ± ± ± ± ± ± ± ± ± 3 5 E CD C B DE B A E DE E C 24 70 93 57 57 117 122 66 28 45 36 ± ± ± ± ± ± ± ± ± ± ± 17 27 11 10 51 64 14 3.8 15 41 12 10 51 61 17 6.3 10 5.2 ± ± ± ± ± ± ± ± ± ± ± 0.5 1 23 32 0.4 0.5 47–51 44–50 43–47 43–47 43–49 40–45 39–41 43–47 43–47 44–50 46–51 °C °C °C °C °C °C °C °C °C °C °C 3.4–4.1 3.4–4.1 3.4–3.8 3.4–4.1 3.6–4.1 3.4–4.1 3.8–3.8 3.5–4.1 3.6–4.1 3.5–4.1 3.4–4.1 reflect 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 wildtype 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 solubilization 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 1102 Optimal conditions 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 especially 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 weakening 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 attributed to these mutations exerting an effect on the FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al C 40 WU-AX solubilisation (% of WU-AX) WU-AX solubilisation (% of WU-AX) A Secondary substrate binding in GH11 xylanases 30 20 10 40 30 20 10 0 30 20 10 10 20 30 40 Enzyme concentration (×10–8 M) 40 30 20 10 10 20 30 40 Enzyme concentration (×10–8 M) LEGEND XBS wild-type XBS W185A XBS G56A-T183A-W185A 10 20 30 40 Enzyme concentration (×10–9 M) D 40 0 OSX solubilisation (% of OSX) OSX solubilisation (% of OSX) B 10 20 30 40 Enzyme concentration (×10–10 M) XBS N54W XBS N54W-N141Q LEGEND XAN wild-type XAN Y29A XAN E31A XAN Y29A-E31A XAN D16A-Y29A-E31A Fig 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 overall 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 Azowheat AX in Table therefore not show clear trends at first sight The ratios of activities on Xylazyme AX over X6, however, confirm the results of the screening Solubilization experiments with the natural 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 especially spectacular on OSX Comparison of XBS and XAN For XBS, it is clear that the residues involved in secondary 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 residues 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 procedure can indeed provide a valuable tool for the initial 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 natural substrates The drop in activity on polymeric substrates upon mutations in the SBS is substantial, FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1103 Secondary substrate binding in GH11 xylanases S Cuyvers et al especially 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 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 Azowheat AX [22], and is a determinant for functionality of xylanases in several applications [23] Table 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 differences 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 characterized with regard to its effect on substrate selectivity However, Moers et al [24] reported a drop in the substrate 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-terminal location of this His-tag suggests a likely interference 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 Therefore, 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 Table gives the overall dissociation constants (Kd) derived from these curves XBS Weakening of the SBS of XBS clearly increases Kd values and therefore lower affinities towards both waterunextractable AX and OSX The differences on OSX are more pronounced than those on water-unextractable AX The G56A-T183A-W185A mutant has a Kd 1104 Table 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 calculated 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 Wild-type N181A T183A W185A G56A-T183A-W185A N54W N141Q N54W-N141Q XAN Wild-type D16A Y29A E31A D32A Y29A-E31A D16A-Y29A-E31A Y29W E31Q E31T D32E 1.00 1.05 1.07 1.31 1.57 1.04 1.01 1.00 ± ± ± ± ± ± ± ± 0.10 0.10 0.08 0.10* 0.09* 0.08 0.14 0.11 1.00 1.03 1.11 0.98 1.08 1.04 1.18 1.04 1.06 1.03 0.94 ± ± ± ± ± ± ± ± ± ± ± 0.09 0.04 0.11 0.06 0.05 0.07 0.08* 0.07 0.06 0.06 0.04 *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 of 25 mgỈmL)1 on water-unextractable AX and 29 mgỈmL)1 on OSX, respectively, compared to 8.8 mgỈmL)1 and 0.4 mgỈmL)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 on water-unextractable AX and 17 mgỈmL)1 13 mgỈmL)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 Kd of 5.3 mgỈmL)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-unextractable AX One possibility is that the attempt to enhance substrate binding was successful for waterunextractable AX, although a higher affinity is not necessarily correlated with a higher activity FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al Secondary substrate binding in GH11 xylanases C 100 Enzyme bound to substrate (%) Enzyme bound to substrate (%) A 80 60 40 20 0 10 20 30 WU-AX (mg·mL) 80 60 40 20 0 10 20 30 WU-AX (mg·mL) 40 40 B 10 20 30 OSX (mg·mL) 40 D 100 Enzyme bound to substrate (%) Enzyme bound to substrate (%) 100 80 60 40 20 0 10 20 30 OSX (mg·mL) LEGEND Wild-type XBS XBS W185A XBS G56A-T183A-W185A 100 80 60 40 20 40 LEGEND Wild-type XAN XAN Y29A XAN E31A XBS N54W XBS N54W-N141Q XAN Y29A-E31A XAN D16A-Y29A-E31A Fig 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) A 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 substrate 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 binding affinity The Kd of wild-type XAN is 24 mgỈmL)1 for water-unextractable AX and 3.8 mgỈmL)1 for OSX The Y29A mutation increases the Kd values to 93 mgỈmL)1 and 41 mgỈmL)1 for water-unextractable AX and OSX, respectively The largest increase is seen for the D16A-Y29A-E31A mutant, with Kd values of 122 mgỈmL)1 and 61 mgỈmL)1 on water-unextractable AX and OSX, respectively The binding of mutants aimed at obtaining an enzyme with an increased substrate binding affinity in the SBS also appeared to be negatively affected However, for most of these FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1105 Secondary substrate binding in GH11 xylanases S Cuyvers et al enzymes, this is not reflected by the activity measurements 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 Kd value 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 correlated However, in XBS, stronger binding does not necessarily 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 proposed 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 substrates 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 water-extractable and waterunextractable) The presence of a SBS was even more beneficial for enzyme activity on water-extractable substrate 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 1106 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 substrate 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 barley a-amylase [29] Whatever the case, the demonstrated 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 secondary 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 Furthermore, 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 suggested 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 modified SBS clearly established that the SBS raises the relative 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 waterunextractable substrate, which is tightly associated with other components in the cell wall matrix FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al The 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 multidomain 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 xylanases 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, Germany) Oligonucleotide primers were obtained from SigmaAldrich 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 at °C After centrifugation (1500 g for 10 min), the residue was boiled for 30 in 20 mL water per milligram 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 at room temperature After centrifugation (11000 g for 30 min), the insoluble fraction was lyophilized Secondary substrate binding in GH11 xylanases E coli TOP10 cells (Invitrogen, Groningen, the Netherlands) were transformed with the modified plasmids (3 lL) by heat shock (30 s at 42 °C) 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 pEXP5CT-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 mgỈL)1 culture 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 (Invitrogen) in accordance with the manufacturer’s instructions More specifically, 500 lL of an overnight culture of transformed cells in ‘yeast extract peptone dextrose medium’ containing 100 lgỈmL)1 Zeocine (InvivoGen Cayla, Toulouse, France) was used for the inoculation of 90 mL of ‘buffered complex medium containing glycerol’ The resulting culture was then grown for 16–20 h at 30 °C under continuous shaking before the cells were harvested by centrifugation (2500 g for min) and transferred to 20 mL of ‘buffered complex medium containing methanol’ for induction of protein expression This culture was then incubated for an additional days at 30 °C and 400 lL of pure methanol was added each day After centrifugation (2500 g for 10 min), the supernatant was collected for further purification (see below) One culture typically yielded 5–15 mg of XAN after purification Protein purification Site-directed mutagenesis Expression plasmid pEXP5-CT-xyna was used for heterologous 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 plasmid was used [32] In both plasmids, a stop codon was incorporated after the last nucleotide encoding for the Cterminal 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 XBS XBS and its mutant variants were purified from the E coli cell lysates with cation exchange chromatography, as previously described by Pollet et al [33] To remove the last contaminating 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 Van- FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1107 Secondary substrate binding in GH11 xylanases S Cuyvers et al dermarliere et al [12] Xylanase containing fractions were pooled and dialyzed against a sodium acetate buffer (200 mm, pH 4.0) XBS and XAN All purified XBS, XAN and mutant variants were free from protein impurities as verified by SDS ⁄ PAGE and silver staining was performed on a PhastSystem Unit (GE Healthcare) (Fig S2) Protein quantification XBS The protein concentration of purified XBS samples was determined by measurement of E280 with a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using molar extinction coefficients calculated with protparam software (http://expasy.org/tools/protparam.html) based on the known amino acid sequence of the different mutants The measurement was performed in triplicate XAN Purified XAN samples had a faint yellow colour as a result of components originating from the expression in P pastoris X33 that interfered with the spectrophotometric protein quantification methods Therefore, protein quantification of the XAN samples was performed by hydrolysis of the protein and subsequent separation and quantification of the resulting single amino acids using high-performance anion exchange chromatography with integrated pulsed amperometric detection A 1.0-mL sample containing 6.0 m HCl, 0.1% phenol, 30 lm norleucine (used as internal standard) and an estimated final protein level of 0.01–0.10 mg was incubated for 24 h at 110 °C for hydrolysis after the head space was flushed with nitrogen gas HCl was removed by evaporation and the obtained residue was dissolved in 1.0 mL of water and analyzed on a Dionex BioLC system (Dionex Corporation, Sunnyvale, CA, USA) as previously described by Rombouts et al [34] The protein concentration was calculated based on the values obtained for alanine, threonine, glycine, valine, phenylalanine and aspartate The analysis, starting from protein hydrolysis, was performed in quintuplicate Activity assays For all activity measurements of XBS and its mutants, a McIlvaine buffer (100 mm citric acid + 200 mm sodium phosphate) (pH 6.0) was used, whereas activity measurements of XAN and its mutants were performed in a McIlvaine buffer at pH 4.0 Both buffers contained 1108 0.50 mgỈmL)1 BSA The incubation time in all assays was 60 and the pre-incubation and incubation temperature was 40 °C The stability of the different purified enzymes in these measurement conditions was tested to avoid erroneous conclusions with respect to differences in activity (Fig S1) The substrates used in these measurements were X6, Xylazyme AX, Azo-wheat AX, water-unextractable AX and OSX X6 is a soluble, linear, oligomeric substrate Xylazyme AX and Azo-wheat AX are polymeric chromophoric AX that are water-unextractable and waterextractable, respectively Water-unextractable AX is a substrate isolated from wheat flour Its water-unextractability is mainly a result of ferulic acid cross-links between AX molecules and interactions with other cell wall components OSX has a low degree of substitution and is considered to be insoluble as a result of partial alignment of unsubstituted regions [35] The water-unextractable AX used in the present study had an average arabinose ⁄ xylose ratio of 0.60 and the average arabinose ⁄ xylose ratio of the OSX was 0.08, as assessed by hydrolysis of the materials and analysis of the noncellulosic monosaccharide content by GC following derivatization, as described previously [36] For measurements on X6, Xylazyme AX and Azo-wheat AX, enzyme dilutions were chosen in the range where a linear correlation between enzyme concentration and hydrolyzed substrate was valid and therefore no substrate exhaustion occurred Activity on X6 After 10 of pre-incubation, 20 lm X6, 37.5 lm rhamnose (used as internal standard) and an appropriate enzyme dilution were mixed in a final volume of 650 lL To ensure measurement at substrate saturation, enzyme dilutions were chosen in such a way that, in the final sample, less than 30% of the X6 was hydrolyzed After 60 of incubation, the reaction was terminated For XBS samples, this was carried out by the addition of 20 lL of NaOH (2.00 m) and subsequent boiling of the sample for 30 XAN samples were simply boiled for 30 Samples were filtered (Millex-GP, 0.22 lm, polyethersulfone; Millipore, Carrigtwohill, Ireland) before separation and quantification of the hydrolysis products by high-performance anion exchange chromatography with integrated pulsed amperometric detection on a CarboPac PA-100 column (250 · mm) performed on a Dionex ICS-3000 system (Dionex Corporation) A linear gradient of 0–125 mm sodium acetate in 100 mm sodium hydroxide over 30 was used for elution (1.0 mLỈmin)1) A standard solution containing xylooligosaccharides (xylose to X6) and rhamnose was used to identify and quantify the hydrolysis products One unit of enzyme activity on X6 (X6U) corresponds to the enzyme concentration needed for the formation of 1.0 lm xylotriose from excess X6 under the conditions of the assay FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS S Cuyvers et al Activity on Xylazyme AX After 10 of pre-incubation, a Xylazyme AX tablet was added to 1.0 mL of an appropriate enzyme dilution The reaction was terminated by the addition of 10.0 mL of Tris solution (1.0% w ⁄ v), vigorous vortex-mixing and immediate filtration E590 of the filtrate was measured One unit of enzyme activity on Xylazyme AX (XyU) corresponds to the enzyme concentration required to obtain E590 = 1.0 after subtraction of the control value (no enzyme) under the conditions of the assay Activity on Azo-wheat AX An appropriate enzyme dilution and liquid Azo-wheat AX substrate were pre-incubated separately for 10 before 500 lL of the substrate was added to 500 lL of enzyme solution The reaction was terminated after 60 by the addition of 2.5 mL of ethanol and vigorous vortex-mixing The mixture was put on ice for 10 before being centrifuged (3000 g for 10 at °C) Then, the E590 of the supernatant was measured One unit of enzyme activity on Azo-wheat AX (AzU) corresponds to the enzyme concentration needed to obtain E590 = 1.0 after subtraction of the control value (no enzyme) under the conditions of the assay and the assumption that no substrate exhaustion occurs Solubilization of water-unextractable AX and OSX Water-unextractable AX isolated from wheat flour and the insoluble fractions isolated from oat spelt xylans were used as substrates To determine the solubilization of the substrates by XBS, XAN and its variants, 20 mg of substrate was incubated with enzyme in a final volume of 7.80 mL after 10 of pre-incubation During incubation, the samples were continuously stirred For OSX, the reaction was terminated by the addition of 230 lL of 4.00 m NaOH and by separating the nonsolubilized from the solubilized material by ltration (MN615; Macherey-Nagel, Duren, Geră many) To avoid alkaline solubilization of waterunextractable AX, the reaction with water-unextractable AX was terminated by first separating the nonsolubilized from the solubilized material by filtration and by then immediately mixing the filtrate with 230 lL of 4.00 m NaOH The monosaccharide content in the filtrate was analyzed by GC following hydrolysis and derivatization, as previously described by Gebruers et al [36] Temperature and pH optima The optimal temperature for enzyme activity under the conditions of the activity measurement assays was determined by measuring activity on Xylazyme AX, under the conditions described above, using different incubation tempera- Secondary substrate binding in GH11 xylanases tures, in the range 30–60 °C at temperature intervals of °C The optimal pH for enzyme activity was determined by measuring activity on Xylazyme AX, under the conditions described above, in McIlvaine buffers with different pH, in the pH range 4.5–7.5 for XBS and 2.5–6.5 for XAN at intervals of 0.5 pH Binding affinity towards insoluble polymers To study the binding affinity of enzymes towards insoluble substrates, 1.0 mL of enzyme solution (91.4 · 10)10 m), diluted in the same buffer as that used for activity measurements, was incubated for 10 on ice with different substrate concentrations The 14 concentrations used varied in the range 0–40.0 mg for water-unextractable AX and 0–30.0 mg for OSX The mixtures were then centrifuged (13000 g for at °C) and the residual activity in the supernatants was measured with Xylazyme AX tablets, as described above For affinity towards water-unextractable AX and OSX, the percentage of bound protein was plotted as a function of the concentration of polymer used for the incubation The obtained graphs were fitted in graphpad prism, version 3.03 (GraphPad Software Inc., San Diego, CA, USA), with a ‘one site binding model’ that results in an overall dissociation constant (Kd): fraction bound protein = (maximum fraction bound protein · [polysaccharide]) ⁄ (Kd + [polysacchaide]) For most measurements, an apparent Kd value is presented because the substrate concentration was a limiting factor in the assay Indeed, at too high substrate concentrations, it is impossible to hydrate all material To evaluate whether the enzymes bind to 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site-directed mutagenesis for the genetic engineering of XBS and XAN This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1111 ... 2011 FEBS S Cuyvers et al Secondary substrate binding in GH11 xylanases Table The effect of genetic engineering of the secondary binding site of the B subtilis and A niger xylanases on the screening... ª 2011 FEBS 1101 Secondary substrate binding in GH11 xylanases S Cuyvers et al Table Biochemical characterization of B subtilis and A niger xylanases with a modified secondary binding site Values... Kd 1104 Table 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