Tài liệu Báo cáo khoa học: The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a b-glycosidase docx

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a b-glycosidase Lu ´ cio M. F. Mendonc¸a and Sandro R. Marana Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil The b-glycosidases from family 1 of the glycoside hydrolases are widely distributed among living organ- isms, being found in bacteria, archea and eukaria. These enzymes are involved in a high diversity of physi- ological roles [1,2]. b-glycosidases catalyze the hydro- lytic removal of the monosaccharide from the non-reducing end of b-glycosides [2,3]. Their active site may be divided into subsites, which are of sufficient size to bind a monosaccharide unit. The monosaccharide forming the non-reducing end of the substrate, called glycone, is bound at subsite )1, whereas the remaining part of the substrate, called aglycone, interacts with the aglycone-binding site, which may be composed of several subsites identified by positive numerals. The substrate is cleaved between subsites )1 and +1 [4]. b-glycosidases are active upon a broad range of sub- strates, as evidenced by a total of 15 different EC numbers grouped in family 1 of the glycoside hydro- lases. Fucose, glucose, galactose, mannose, xylose, 6-phospho-glucose and 6-phospho-galactose are recog- nized by the b-glycosidase subsite )1. Nevertheless, the diversity of aglycones is higher, including monosaccha- rides, oligosaccharides and aryl and alkyl moieties [2]. Furthermore, the aglycone specificity is an important factor for determining the physiological functions of b-glycosidases. Keywords aglycone; catalysis; glycoside hydrolase; specificity; b-glycosidase Correspondence S. R. Marana, Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, Universidade de Sa˜o Paulo, CP 26077, Sa˜o Paulo, 05513-970 SP, Brazil Fax: +55 11 3815 5579 Tel: +55 11 3091 3810 E-mail: srmarana@iq.usp.br (Received 1 February 2008, revised 4 March 2008, accepted 13 March 2008) doi:10.1111/j.1742-4658.2008.06402.x The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a b-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investi- gated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contrib- ute to stabilize ES à . Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding. Abbreviations BglB, b-glycosidase from Paenibacillus polymyxa; SbDhr1, b-glycosidase from Sorghum bicolor;Sfbgly, b-glycosidase from Spodoptera frugiperda; ZmGlu1, b-glycosidase from Zea mays. 2536 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS Structural studies of complexes between b-glycosid- ases and substrates or inhibitors revealed residues that are present in subsite )1 and interact through hydro- gen bonds with the glycone hydroxyls. In addition, the role of these interactions and residues in determining glycone specificity has been characterized through site-directed mutagenesis, enzyme kinetics and bio- energetics [5–15]. Previous studies using oligocellodextrins showed that diverse b-glycosidases have a different number of subsites forming their aglycone-binding sites [15]. Nevertheless, just recently the structures of b-glyco- sidases (ZmGlu1 from Zea mays, SbDhr1 from Sorghum bicolor and BglB from Paenibacillus poly- myxa) containing ligands in their aglycone-binding site were obtained [13,16,17]. These data revealed that the subsite +1 is formed by two walls that embrace the aglycone moiety. Subsites +1 of ZmGlu1 and BglB are narrow, whereas subsite +1 of SbDhr1 has a wider opening [13,17,18]. One of these walls (also called the basal platform) is formed by the side chain of a conserved tryptophan (W378 in ZmGlu1, W376 in SbDhr1 and W328 in BglB), whereas the residues forming the other wall (also called the ceiling) are highly variable among these enzymes. In ZmGlu1 the subsite +1 is formed by bulky and apolar amino acid residues T194, F198, F205 and F466, whereas T192, V196, L203 and S462 are found in subsite +1 of SbDhr1 [13]. These residues correspond to C170, L174, H181 and A411 in BglB, which, together with Y169, N223, E225, Q316 and W412, form subsite +1. Data from the BglB–cellotetraose complex also revealed the residues forming subsites +2 and +3 [13]. The type of residues forming subsite +1 of ZmGlu1 indicate a prevalence of hydrophobic interac- tions with the aglycone, which are interactions that present less restriction about the positioning of its participant. Hence, this could explain the broad agly- cone specificity of ZmGlu1. On the other hand, the presence of hydrogen bond-forming residues would determine a more restricted aglycone positioning in SbDhr1, which is only active on dhurrin [13,16]. In subsite +1 of BglB, which is active on oligocellodext- rins, in spite of the presence of hydrophobic residues L174 and A411, a network of hydrogen bonds hold glucose units at subsites +1, +2 and +3 [17]. Therefore, in addition, to facilitate the identifica- tion of amino acid residues composing the aglycone- binding site, the structural data of b-glycosidase complexes have been used to infer the molecular basis of the specificity for aglycone. Indeed, the balance between hydrophobic interactions and hydrogen bonds seems to be important for the aglycone speci- ficity. However, this model, which is a general description of the interactions involved in the recog- nition of aglycone, is not complete, as demonstrated by the site-directed mutagenesis experiments intending to exchange the aglycone specificity between SbDhr1 and ZmGlu1 [15,19]. Therefore, this model should be developed to establish the contribution of each resi- due in the aglycone-binding site to the binding of diverse types of aglycones. Furthermore, these data may be of particular importance in understanding the physiological role of b-glycosidases and in designing inhibitors. In addition, another important issue in understand- ing the aglycone specificity is the contribution of the interactions with the aglycone to the catalysis in b-glycosidases. Mutational studies with ZmGlu1 and SbDhr1 indicate that residues forming subsite +1 con- tribute to the catalysis, probably by stabilizing the ES à complex of the glycosylation step [15,19]. Besides that, structural data from the complexes of ZmGlu1 and SbDhr1 with their natural substrates (dimboa-glc and dhurrin, respectively) and an inhibitor (glucotetrazole) suggested that the interactions with the aglycone could affect the glycone positioning within subsite )1 [13]. Nevertheless, details about the role of different resi- dues of the aglycone-binding site in the stabilization of ES à and the interdependence between the binding of aglycone and the positioning of glycone in subsite )1 are not known. In this study, the role in the substrate specificity and catalysis of four residues (E190, E194, K201 and M453) forming the aglycone-binding site of a digestive b-glycosidase from Spodoptera frugiperda (fall army- worm) (Sfbgly; EC 3.2.1.21; GenBank accession no.: AF052729) was evaluated through site-directed muta- genesis and enzyme kinetic experiments. The roles of E190, E194, K201 and M453 in aglycone binding were characterized using competitive inhibitors with different types of aglycones (oligosaccharides, and alkyl and phenyl moieties), whereas p-nitrophenyl b-glycosides (fucosides, glucosides, galactosides and xylosides) were used to evaluate the catalytic contri- bution of E190, E194, K201 and M453. Sfbgly, which is classified in family 1 of the glycoside hydrolases, has an active site composed of four subsites ()1, +1, +2 and +3) [20]. The interactions formed between the substrate glycone and residues Q39 and E451, which are part of the Sfbgly active site, explain the preference of this enzyme for b-glucosides, b-galacto- sides and b-fucosides [10,14]. Nevertheless, the molec- ular basis of the broad aglycone specificity of Sfbgly had not been studied previously. L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2537 Results and Discussion Role of residues E190, E194, K201 and M453 in aglycone binding The spatial structures of ZmGlu1, SbDhr1 and BglB revealed groups of amino acid residues that formed the binding site of the substrate aglycone. These aglycone- binding sites share a common structural element, a basal platform formed by a tryptophan residue (W378 in ZmGlu1, W376 in SbDhr1 and W328 in BglB), but they also have a variable portion (called the ceiling). This portion is formed by T194, F198, F205 and F466 in ZmGlu1, and by V196, L203 and S462 in SbDhr1. These residues correspond to C170, L174, H181 and A411 in BglB, which, together with Y196, N223, E225, Q316 and W412, form the aglycone-binding site (Fig. 1). Hence, these residues are potentially involved in aglycone binding [13,17]. In addition, other residues form a ‘layer’ that helps in the positioning of the resi- dues that interact directly with the aglycone (basal platform and ceiling) [15]. Nevertheless, the relative contributions of the aglycone-binding residues in the substrate binding and catalysis are not known for any b-glycosidase. Sequence alignment and structural comparison indi- cated that residues E190, E194, K201 and M453, which correspond to T194, F198, F205 and F466 in ZmGlu1, may be part of the aglycone-binding site of Sfbgly (Fig. 1). Thus, in order to establish the relative contribution of these residues to the interaction with different types of aglycone, they were replaced through site-directed mutagenesis, generating the mutants E190A, E190Q, E194A, K201A, K201F and M453A. Replacements with A were made to remove side chains that could interact with the aglycone. Mutations E190Q and K201F were planned taking into account the residues found in the b-glycosidase from A B E D C Fig. 1. Comparison of the aglycone-binding site of some b-glycosidases. (A) Aglycone-binding site of ZmGlu1. The aglycone and glycone of the substrate (dimboa-Glc) are also indicated. (B) Aglycone-binding site of Sfbgly. Residues E190, E194, K201 and M453 are structurally equivalent to T194, F198, F205 and F466. The substrate (dimboa-Glc) was included to facilitate the visualization of the active site. (C) Agly- cone-binding site of SbDhr1, including the substrate dhurrin. (D) Aglycone-binding site of BglB, including the inhibitor thiocellobiose. (E) Sequence alignment of some b-glycosidases showing residues (boxes) forming the aglycone-binding site. Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana 2538 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS Tenebrio molitor (AF312017) [21], which is closely related to Sfbgly (45% identity; 65% similarity). These insect b-glycosidases were previously characterized and showed differences in their aglycone specificity [20,21]. The mutant enzymes were expressed in bacteria and purified through hydrophobic chromatography (sup- plementary Fig. S1). Then, dissociation constants (K i ) for the complex between these mutant enzymes and different competitive inhibitors were determined and compared with those from the wild-type Sfbgly (Table 1). A series of alkyl b-glucosides (hexyl b-glucoside to nonyl b-glucoside) was initially used to characterize the Sfbgly mutants (Table 1). Molecular models of these alkyl b-glucosides indicate that each four methy- lene groups of their aglycone may cover an area simi- lar to that of one glucose unit. Thus, this series of alkyl b-glucosides is useful for probing mutational effects along the aglycone-binding site. Based on the K i values (Table 1), the binding energy (DG 0 ) for the pentyl moiety formed by the methylene groups 2 to 6 of an alkyl-type aglycone was calculated by subtracting the DG 0 for methyl b-glucoside from that for hexyl b-glucoside. These data indicated a favorable binding (DG 0 = )2.9 kJÆmol )1 ) between the pentyl moiety and the wild-type Sfbgly, whereas the energy of that interaction was reduced by mutations E194A, K201A and M453A. Mutation K201F did not affect the binding of that aglycone moiety (Fig. 2). However, the binding of the pentyl moiety in the agly- cone-binding site was unfavorable for the mutants E190A (DG 0 = +3.5 kJÆmol )1 ) and E190Q (DG 0 = +3.3 kJÆmol )1 ) (Fig. 2). Indeed, these mutations caused the most drastic effects on the binding of the initial pentyl moiety (converting a favorable interaction to an unfavorable one). In addition, the binding energy of the terminal portion of the alkyl-type aglycone (from the seventh to the ninth methylene group) was determined by subtracting the D G 0 for hexyl b-gluco- side from that for nonyl b -glucoside (Fig. 2). The favorable interaction (DG 0 = )4.9 kJÆmol )1 ) observed for the wild-type Sfbgly was not significantly affected by any of the mutations, except for K201F, which increased the energy of that interaction (DG 0 = )8.1 kJÆmol )1 ). Table 1. Inhibition data for the wild-type and mutant Sfbgly. All inhibitors were simple linear competitive. K i values were calculated using ENZFITTER software. The data were obtained with at least five different concentrations of substrate (methylumbeliferyl b-glucoside) in the presence of at least five different concentrations of inhibitor. heptylbglc, heptyl b-glucoside; hexylbglc, hexyl b-glucoside; methylbglc, methyl b-glucoside; nonylbglc, nonyl b-glucoside; octylbglc, octyl b-glucoside; phenylbglc, phenyl b-glucoside; wt, wild-type. Enzyme E190A E194A K201A M453A K201F E190Q wt Inhibitor K i (mM) Phenylbglc 30 ± 2 36 ± 1 74 ± 4 52 ± 1 48 ± 1 78 ± 1 49 ± 2 Methylbglc 8.0 ± 0.2 10.1 ± 0.1 15.6 ± 0.4 10.2 ± 0.4 10 ± 1 12.1 ± 0.3 5.1 ± 0.7 Cellobiose 17 ± 1 1.2 ± 0.1 81 ± 3 5.2 ± 0.3 30 ± 1 13.7 ± 0.4 2.9 ± 0.3 Cellotriose 2.3 ± 0.2 0.070 ± 0.004 0.41 ± 0.01 0.09 ± 0.01 0.04 ± 0.01 8.6 ± 0.3 0.27 ± 0.01 Cellotetraose 2.4 ± 0.1 0.14 ± 0.01 0.54 ± 0.03 0.21 ± 0.01 0.25 ± 0.03 19.6 ± 0.7 0.20 ± 0.02 Cellopentaose 2.0 ± 0.1 0.20 ± 0.01 2.3 ± 0.1 0.26 ± 0.01 0.30 ± 0.02 16.2 ± 0.3 0.25 ± 0.02 Hexylbglc 32 ± 2 8.0 ± 0.4 10.0 ± 0.6 5.6 ± 0.2 3.3 ± 0.2 44.9 ± 0.7 1.57 ± 0.05 Heptylbglc 21 ± 1 3.6 ± 0.1 4.2 ± 0.2 2.6 ± 0.1 1.12 ± 0.06 21.8 ± 0.5 0.7 ± 0.1 Octylbglc 10.3 ± 0.6 1.49 ± 0.03 2.52 ± 0.09 1.30 ± 0.06 0.42 ± 0.03 11.0 ± 0.2 0.44 ± 0.02 Nonylbglc 5.9 ± 0.2 0.83 ± 0.02 1.16 ± 0.04 0.50 ± 0.03 0.13 ± 0.01 5.7 ± 0.3 0.22 ± 0.03 Fig. 2. Binding energies for the interactions between different types of aglycone and the wild-type and mutant Sfbgly. Negative values correspond to favorable interactions, whereas positive val- ues represent unfavorable binding. Black bars indicate an aglycone formed by a phenyl moiety; white bars indicate the initial portion of an alkyl-type aglycone (pentyl moiety formed by methylene groups 2 to 6); and gray bars indicate the terminal portion of an alkyl-type aglycone (moiety formed by methylene groups 7 to 9). wt, wild-type. L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2539 Therefore, these results indicate that residues E190, E194, K201 and M453 are part of the aglycone-bind- ing site of Sfbgly and interact with the initial pentyl moiety formed by methylene groups 2 to 6 of the alkyl-type aglycones. On the other hand, the binding of alkyl moieties in the more external portion of the aglycone-binding site, which is occupied by methylene groups 7 to 9, is not influenced by those residues. The binding energies for an aglycone formed by a phenyl moiety were calculated using the K i values for phenyl b-glucoside and methyl b-glucoside (Table 1). These data showed an unfavorable binding of the phe- nyl-type aglycone with the wild-type and all-mutant Sfbgly (Fig. 2). Mutations E190 and E194A resulted in only small reductions (about 40%) of that unfavorable interaction, but they did not convert it to a favorable binding. Thus, by contrast with the binding of alkyl- type aglycones, single mutations were unable to cause large changes in the interaction of phenyl-type agly- cones with Sfbgly. In addition to the analysis of the interaction with alkyl-type and phenyl-type aglycones, the dissociation constants (K i ) of the complexes between oligocellodext- rins and the wild-type and mutant Sfbgly were also determined (Table 1). Based on these results, the bind- ing energies for each glucose unit of the aglycone (DG 0 ⁄ glucose unit) were calculated (Fig. 3). For orien- tation purposes, the glucose unit forming the non-reducing end of the aglycone was called the ‘first glucose unit’ and the other glucose units were sequen- tially named in the direction of the reducing end of the aglycone. Thus, mutations E190A, E194A, K201A and K201F affected the binding of the first glucose unit of the aglycone (DDG 0 = +3.3, )3.9, +5.5, +4.1 kJÆmol )1 , respectively), whereas M453A did not influence that interaction. Mutations M453A, K201A and K201F increased the affinity for the second glucose unit of the aglycone (DDG 0 = )4.2, )7.3, )10 kJÆmol )1 , respec- tively), whereas E190A and E194A did not affect that interaction. It is noteworthy that E190 and E194 inter- act with the first glucose unit of the aglycone, whereas M453 interacts with the second glucose unit of the aglycone. However, K201 interacts with both glucose units. In addition, mutations E194A, K201A, K201F and M453A also significantly affected the binding of the third glucose unit of the aglycone (DDG 0 = +2.5, +1.4, +5.3, +2.8 kJÆmol )1 , respectively). The fourth glucose unit of aglycone is not bound by any mutant sfbgly. The same result has been previously observed for the wild-type sfbgly. The exception to these trends is the mutant E190Q, which showed a very low affinity for any glucose unit of the aglycone. Previously, it has been proposed that each glucose unit of the aglycone interacts with a specific portion of the aglycone-binding site, which was named subsite [4]. Taking into account this definition and combining the results presented above it may be proposed that resi- dues E190 and E194 are positioned in an internal region of the aglycone-binding site, probably subsite +1, because they influence the binding of the first glu- cose unit of the aglycone. On the other hand, M453 may be positioned in subsite +2 because these residues influence the binding of the second glucose unit of the aglycone. Residue K201 may be part of both subsites +1 and +2 or may be located in the interface between them as it simultaneously affects the binding of the first and the second glucose unit of the aglycone. This proposal is in agreement with the binding data for alkyl-type aglycones (Fig. 2), because the pentyl moiety formed by the methylene groups 2 to 6 is large enough to fill subsite +1 and to occupy part of subsite +2. As a result, residues E190, E194, K201 and M453 can still interact with that initial portion of an alkyl-type aglycone, even though they interact with different units of an oligosaccharidic aglycone. Figure 3 also showed that mutations K201A and K201F affected the interactions with the first three glu- cose units of the aglycone, whereas M453A caused modifications in the interaction with the second and third glucose units. This suggests that modifications in the interactions with a specific glucose unit of the agly- cone may alter the conformation and ⁄ or freedom of the other glucose units, which are part of the aglycone, affecting its interactions with the aglycone site. Fig. 3. Binding energies for the interactions between glucose units of the aglycone and the wild-type and mutant Sfbgly. Negative values represent favorable interactions, whereas positive values correspond to unfavorable binding. Glucose units were numbered from the non-reducing to the reducing end of the aglycone. wt, wild-type. Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana 2540 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS Following the characterization of the binding of different types of aglycone, a comparative analysis of the mutational effect on their binding revealed that residues E190, E194, K201 and M453 have different roles in the determination of the Sfbgly specificity for the substrate aglycone. Thus, although the bulky and apolar phenyl moieties may form favorable interac- tions with the basal platform (W378 in Sfbgly), the binding of these moieties is dominated by unfavorable interactions with the polar residues E190, E194 and K201, whereas M453 has little influence on this. The replacement of E190, E194 and K201 with A, which has a small side chain, reduced those unfavorable interactions (Fig. 2), and, interestingly, the more com- pact alkyl moieties overcame those unfavorable inter- actions. Indeed, residues E190, E194, K201 and M453 contributed to the favorable binding of the initial pen- tyl moiety of the aglycone. Of these residues, E190 is the most important for that interaction because muta- tions E190A and E190Q resulted in the largest decrease of affinity for the pentyl moiety (DDG 0 = 6.4 and 6.2 kJÆmol )1 , respectively); E194, K201 and M453 contribute similarly to the binding of pentyl moieties given that the replacement of those residues by A resulted in similar decrease (when taking into account the experimental errors) of the affinity (DDG 0 $ 2kJÆmol )1 ) for pentyl moieties. However, these same residues give different contributions to the binding of the glucose units of the aglycone. Thus, M453 does not affect the binding of the first glucose unit (DDG 0 $ 0; Fig. 4); in fact, it interacts with the second glucose unit of the aglycone (Fig. 3). E194 has an unfavorable interaction with the first glucose unit, given that replacement of E194 with A increased, by 3.9 kJÆmol )1 , the affinity for that glucose unit (Fig. 4). Conversely, E190 and K201 may form interactions (probably hydrogen bonds) that are important for the binding of the first glucose units of the aglycone because their replacement with A caused a large decrease in the affinity for glucose (DDG 0 = +3.9 and +5.6 kJÆmol )1 , respectively; Fig. 4). In agreement, K201 corresponds to H181 in BglB, a residue that inter- acts through a hydrogen bond with glucose units at sub- site +1 of that enzyme [17]. Moreover, E190 corresponds to T194 in ZmGlu1, which may form a hydrogen bond with the aglycone of dimboa-Glc. It is noteworthy that, regarding aglycone binding, mutation K201F drastically reduced the glucose binding without affecting the affinity for alkyl moieties. In addi- tion, mutation K201F also increased the affinity for the terminal portion of alkyl-type aglycones (Fig. 2). Resi- due K201 is replaced by F in the b-glycosidases from Tenebrio molitor (Tmbgly; AF312017) and Cavia porce- lus (Cpbgly; U50545). Moreover, F179 is part of the aglycone-binding site of Cpbgly [22]. In order to com- pare the aglycone-binding sites of these b-glycosidases, the effect of the aglycone size on the binding of several alkyl b-glucosides was measured. Interestingly, these binding data showed that each aglycone methylene moiety of the aglycone increases in 1.6 kJÆmol )1 the affinity of the wild-type Sfbgly for alkyl b-glycoside (Fig. 5), whereas mutation K201F increased that parameter to 2.6 kJÆmol )1 , which was similar to that of Cpbgly (3.0 kJÆmol )1 ) and higher than that of Tm bgly (1.0 kJÆmol )1 ) [21,23]. Therefore, residues in posi- tion 201 (and its equivalent in other b-glycosidases) may be an important factor in the determination of sub- site +1 preference for alkyl-type aglycones. In summary, the triad formed by E190, E194 and K201 controls the specificity for the substrate Fig. 4. Mutational effect (DDG 0 ) on the binding energy of alkyl moi- eties and glucose units of the aglycone. Black bars correspond to alkyl moieties and white bars correspond to glucose units. Positive DDG 0 values correspond to a decrease in affinity, whereas negative DDG 0 values represent an increase in affinity. –10 –15 –20 –25 98 7 6 5 Aglycone carbon number Binding energy (kJ·mol –1 ) Fig. 5. Effect of the aglycone size on the binding between alkyl b- glucosides and the wild-type and mutant Sfbgly. ( ), wild-type; ( ), mutant K201F. L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2541 aglycone. This triad reduces the affinity for phenyl moieties. Residue E190 drives the binding of both alkyl moieties (the initial portion up to the sixth methylene group) and the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the specificity for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interac- tion with alkyl moieties. Therefore, the replacement of E194 and K201 may be an important mechanism for changing the specificity of Sfbgly for aglycone. The relative contribution to aglycone specificity of residues corresponding to E190, E194, K201 and M453 is probably different in other b-glycosidases, especially in view of the broad variety of residues occupying those positions. Nevertheless, the differen- tial participation of several residues in the definition of the aglycone specificity may be a general trend. Catalytic role of residues E190, E194, K201 and M453 from the Sfbgly aglycone-binding site Steady-state kinetic parameters for the hydrolysis of p-nitrophenyl b-glycosides and methylumbelliferyl b-glucosides by the wild-type and mutant Sfbgly were determined (Table 2). In order to evaluate the role of residues E190, E194, K201 and M453 on the catalytic Table 2. Steady-state kinetic parameters for the hydrolysis of p-nitrophenyl b-glycosides and methylumbeliferyl b-glucoside by wild-type and mutant Sfbgly. Experiments were carried out using at least 10 different substrate concentrations. Parameters were calculated using ENZFITTER software. MUbglc, methylumbeliferyl b-glucoside; NPbfuc, p-nitrophenyl b-fucoside; NPbgal, p-nitrophenyl b-galactoside; NP bglc, p-nitrophe- nyl b-glucoside; NPbxyl, p-nitrophenyl b-xyloside. Enzyme Substrate K m (mM) k cat (s )1 ) k cat ⁄ K m (s )1 ÆmM )1 ) Relative k cat ⁄ K m (%) E190A NPbfuc 0.59 ± 0.05 0.348 ± 0.009 0.58 ± 0.05 100 NPbglc 1.05 ± 0.05 0.59 ± 0.01 0.56 ± 0.02 90 NPbgal 8.2 ± 0.2 0.072 ± 0.001 0.0087 ± 0.0002 1.5 NPbxyl 4.9 ± 0.4 0.00188 ± 0.00009 0.00038 ± 0.00003 0.06 MUbglc 6.3 ± 0.6 0.020 ± 0.001 0.0031 ± 0.0003 0.5 E194A NPbfuc 0.38 ± 0.03 0.98 ± 0.01 2.5 ± 0.2 100 NPbglc 1.20 ± 0.07 0.56 ± 0.01 0.46 ± 0.02 18 NPbgal 9.0 ± 0.3 0.189 ± 0.003 0.0210 ± 0.0007 0.8 NPbxyl 4.8 ± 0.2 0.0127 ± 0.0003 0.0026 ± 0.0001 0.1 MUbglc 2.6 ± 0.1 0.2674 ± 0.0004 0.102 ± 0.003 3.9 K201A NPbfuc 0.71 ± 0.05 2.43 ± 0.03 3.4 ± 0.2 100 NPbglc 2.6 ± 0.2 0.66 ± 0.02 0.25 ± 0.02 7.5 NPbgal 4.9 ± 0.3 0.058 ± 0.001 0.0118 ± 0.0007 0.3 NPbxyl 2.4 ± 0.1 0.0073 ± 0.0001 0.0030 ± 0.0001 0.09 MUbglc 2.6 ± 0.2 0.032 ± 0.001 0.0123 ± 0.0001 0.3 M453A NPbfuc 0.65 ± 0.04 4.15 ± 0.05 6.3 ± 0.4 100 NPbglc 1.26 ± 0.03 1.52 ± 0.01 1.20 ± 0.02 18 NPbgal 4.4 ± 0.2 0.174 ± 0.004 0.039 ± 0.002 0.6 NPbxyl 3.6 ± 0.3 0.0238 ± 0.0009 0.0066 ± 0.0006 0.1 MUbglc 1.7 ± 0.1 0.62 ± 0.01 0.36 ± 0.02 5.7 K201F NPbfuc 0.62 ± 0.06 8.9 ± 0.1 14 ± 1 87 NPbglc 1.2 ± 0.1 4.0 ± 0.1 3.3 ± 0.2 20 NPbgal 6.2 ± 0.3 4.03 ± 0.09 0.65 ± 0.03 3.9 NPbxyl 1.04 ± 0.06 0.089 ± 0.001 0.085 ± 0.005 0.5 MUbglc 0.08 ± 0.01 1.32 ± 0.08 16 ± 2 100 E190Q NPbfuc 0.66 ± 0.04 0.668 ± 0.009 1.00 ± 0.06 100 NPbglc 2.10 ± 0.09 0.65 ± 0.01 0.30 ± 0.01 30 NPbgal 9.9 ± 0.5 0.153 ± 0.003 0.0154 ± 0.0008 1.5 NPbxyl 4.7 ± 0.4 0.0211 ± 0.0008 0.0044 ± 0.0004 0.4 MUbglc 6.2 ± 0.4 0.114 ± 0.004 0.018 ± 0.001 1.8 wt NPbfuc 0.67 ± 0.03 7.14 ± 0.06 10.6 ± 0.4 100 NPbglc 0.90 ± 0.04 2.26 ± 0.03 2.5 ± 0.1 23 NPbgal 2.9 ± 0.1 0.269 ± 0.003 0.092 ± 0.003 0.9 NP bxyl 1.56 ± 0.05 0.0302 ± 0.0003 0.0193 ± 0.0006 0.02 MUbglc 2.3 ± 0.1 0.231 ± 0.003 0.100 ± 0.004 0.9 Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana 2542 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS steps, these data were used to calculate the effect of mutations of these residues on the stability of the ES à complex for p-nitrophenyl b-glucoside, p-nitrophenyl b-fucoside, p-nitrophenyl b-galactoside and p-nitrophe- nyl b-xyloside hydrolysis. Figure 6 shows that all mutations destabilized the ES à complex for all sub- strates tested, except mutation K201F, which resulted in stabilization of the ES à complex for p-nitrophenyl b-galactoside and p-nitrophenyl b-xyloside hydrolysis. Destabilization of ES à indicates a reduction in the rate of substrate hydrolysis, whereas the opposite is valid for the stabilization of ES à . Hence, the existence of these mutational effects on ES à stability indicate that residues E190, E194, K201 and M453 participate in the catalysis. Considering that the catalytic mechanism of b-glycosidases is divided into two steps (glycosyla- tion and deglycosylation), and that the aglycone is released in the first step [2], the interactions of these residues with the aglycone probably contribute to sta- bilizing the ES à complex of the glycosylation step. Interestingly, a comparison of these mutational effects with those resulting from the mutation of resi- dues Q39 and E451, which are involved in the glycone binding in Sfbgly [10], showed that the ‘ES à destabiliz- ing effects’ resulting from mutations of the aglycone- binding residues are usually smaller than those from mutations at subsite )1 (Q39A and E451A) (Fig. 6). Nevertheless, the total effect of the mutations of the aglycone-binding residues (20, 20 and 16 kJÆmol )1 for p-nitrophenyl b-fucoside, p-nitrophenyl b-glucoside and p-nitrophenyl b-galactoside hydrolysis, respec- tively) is similar to the combined effect of mutations Q39A and E451A (33, 31 and 32 kJÆmol )1 for p-nitro- phenyl b-fucoside, p-nitrophenyl b-glucoside and p-nitrophenyl b-galactoside hydrolysis, respectively). Hence, the energy of the non-covalent interactions available to stabilize ES à tend to be concentrated in a few residues in subsite )1 (for instance Q39 and E451), whereas these ‘ES à -stabilizing’ interactions are more homogeneously distributed among the aglycone-bind- ing residues. Besides, as the contributions of the agly- cone-binding residues to the stability of ES à are relevant, they should be considered in the design of b-glycosidase inhibitors. Figure 6 also shows that the mutational effects (DDG à ) tend to be similar regardless of the substrate when considering the aglycone-binding residues. This trend is not observed for mutations of residues (Q39 and E451) directly involved in the binding of glycone, or for mutation K201F. Taking into account that the substrates share a common aglycone, but differ in the glycone, the results obtained for the mutants E190A, E194A, K201A and M453A are those expected for res- idues that interact with the substrate aglycone but do not participate in the binding of the substrate glycone. Therefore, an implication of these results is that the interactions with aglycone do not affect the binding of the glycone within the Sfbgly active site. This hypothesis was further investigated by deter- mining the influence on the glycone binding of the Sfbgly interactions with the aglycone. Thus, the effect of the alteration of the glycone structure in the stabil- ity of the ES à complex (DDG à ) for the wild-type and mutant Sfbgly was determined using the k cat ⁄ K m for the hydrolysis of p-nitrophenyl b-glucoside, p-nitrophe- nyl b-fucoside and p-nitrophenyl b-galactoside. These DDG à values represent the manner in which the inter- actions within subsite )1 are affected by the modifica- tion of the glycone structure, in particular the spatial positioning of its hydroxyl groups. Hence, two b-glyco- sidases, presenting exactly the same interaction pattern within subsite )1, should be equally affected by the alteration of the substrate glycone generating the same DDG à . Thus, a plot of these DDG à values for a pair of b-glycosidases presenting identical subsites )1 would be a line showing a correlation coefficient and slope equal to 1. Indeed, comparison between wild-type and mutant Sfbgly using such plots revealed correlation coefficients and slopes close to 1 for all mutants except K201F, which presented a correlation coefficient of 0.58 (Table 3). These results indicate that despite the mutations in the aglycone-binding site, the interactions with the glycone remained very similar for the mutants of Sf bgly analyzed, except for K201F. Therefore, at least for the Sfbgly activity upon p-nitrophenyl b-gly- cosides, the interactions with aglycone do not affect glycone binding. In addition, the catalytic contribu- tions of residues E190, E194, K201 and M453 result exclusively from interactions with the aglycone that stabilize ES à . Conversely, mutation K201F influenced the interaction pattern of the p-nitrophenyl b-glyco- Fig. 6. Mutational effect on the stability of ES à (DDG à ) involving dif- ferent substrates. Dark gray bars, p-nitrophenyl b-fucoside; white bars, p-nitrophenyl b-glucoside; black bars, p-nitrophenyl b-galacto- side; light gray bars, p-nitrophenyl b-xyloside. L. M. F. Mendonc¸a and S. R. Marana Molecular basis for specificity of a b-glycosidase FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS 2543 sides within subsite )1 through modifying the interac- tions with their aglycones, suggesting that in mutant K201F the binding of glycone and aglycone are inter- dependent. These results are not observed for mutant K201A, revealing that such mutational effects are related to the type of residue occupying position 201. Interestingly, the residue corresponding to K201 in BglB is H181, which delineates a channel that could be the pathway for the release of the aglycone in the glyco- sylation step and the entrance of the water molecule involved in the hydrolysis of the covalent intermediate [17]. Thus, the unusual effects of the K201F mutation on the Sfbgly catalysis could be the result of a drastic alteration in that putative channel, which would change the rate of the glycosylation and deglycosylation steps. Materials and methods Site-directed mutagenesis Site-directed mutagenesis experiments were performed as described in the instructions of the kit ‘QuikChange site- directed mutagenesis’ (Stratagene, La Jolla, CA, USA) using a plasmid pT7-7 [24] encoding the wild-type Sfbgly as the template. A pair of mutagenic primers was used to produce each desired mutation. Only the primers corresponding to the sense strand are listed, as follows: mutation E190A, 5¢-caacgagcctagagcgatttgctttgagg-3¢; mutation E190Q, 5¢-caa cgagcctagacagatttgctttgagg-3¢; mutation E194A, 5¢-gagagattt gctttgcgggttatggatctgc-3¢; mutation K201A, 5¢-gttatggatctgct accgcggctccgatcctaaacg-3¢; mutation K201F, 5¢-ggttatggatctg ctacttcgctccgatcctaaacgc-3¢; and mutation M453A, 5¢-ggacaa ctttgaatgggcggagggttatattgag-3¢. The incorporation of muta- tions was verified by DNA sequencing. Expression of recombinant Sfbgly BL21 DE3 cells (Novagen, Darmstadt, Germany) were transformed with pT7-7 plasmids encoding the wild-type and mutant Sfbgly. Transformed bacteria were cultured (37 °C, 150 r.p.m.) in 500 mL of Luria–Bertani (LB) broth containing carbenecillin (50 lgÆmL )1 ) until an attenuance (D) of 0.6–0.8 at 600 nm was reached. Then, the production of recombinant Sfbgly was induced (25 °C, 6 h, 150 r.p.m.) by adding 1 mm isopropyl thio-b-d-galactoside. The induced bacteria were harvested by centrifugation (7000 g, 30 min, 4 °C) and resuspended in 50 mm Hepes buffer containing 150 mm NaCl, 0.02% (w ⁄ v) of hen egg-white lysozyme and 0.1% (v ⁄ v) of Triton X-100. This suspension was incubated at room temperature for 45 min with slow shaking at 30 r.p.m. Then, the suspension was exposed to four pulses (45 s each) of ultrasound using a Branson soni- fier (at output 4.0) adapted with a microtip. Cell debris was harvested by centrifugation (7000 g,4°C, 30 min) and the supernatant was sequentially filtered through cheesecloth and 0.22-lm Millex filters (Millipore, Billerica, MA, USA). Purification of recombinant Sfbgly The soluble material resulting from the lysis of the induced bacteria was mixed with 200 mm sodium phosphate (pH 7.0) containing 3.4 m (NH 4 ) 2 SO 4 (2 : 1, v ⁄ v). This mix- ture was incubated at 4 °C, without shaking, for 16h. The precipitated material obtained after centrifugation (7000 g, 4 °C, 30 min) was discarded,and the supernatant was filtered through cheesecloth and 0.22-lm Millex filters (Millipore). Then, this supernatant was loaded onto a Resource ETH column (GE HealthCare, Chalfont, St Giles, UK). Non-retained proteins were eluted with 50 mm sodium phosphate (pH 7.0) containing 1.27 m (NH 4 ) 2 SO 4 , whereas retained proteins were eluted using a linear gradi- ent of (NH 4 ) 2 SO 4 (from 1.27 to 0 m) prepared in 50 mm sodium phosphate (pH 7.0). Fractions of 1.0 mL were col- lected and analyzed for b-glycosidase activity using 4 mm p-nitrophenyl b-fucoside prepared in 50 mm citrate phos- phate buffer (pH 6.0). Fractions containing b-glycosidase activity were pooled, mixed with (NH 4 ) 2 SO 4 as described above and then loaded onto a Resource ISO column (GE HealthCare). Non- retained proteins were eluted with 50 mm sodium phos- phate (pH 7.0) containing 0.95 m (NH 4 ) 2 SO 4 , whereas retained proteins were eluted using a linear gradient of (NH 4 ) 2 SO 4 (from 0.95 to 0 m) prepared in 50 mm sodium phosphate (pH 7.0). Fractions of 1.0 mL were collected and analyzed for b-glycosidase, as previously described. To ascertain the purity of Sfbgly, fractions containing b-glyco- sidase activity were pooled and submitted to SDS-PAGE analysis followed by silver staining [25,26]. The protein concentrations were determined by using absorbance at 280 nm in the presence of 6 m guanidium hydrochloride prepared in sodium phosphate (pH 6.5). Extinction coefficients (e 280 nm ) were calculated based on the mutant Sfbgly sequences [27,28]. Enzyme kinetic analysis The initial rate of hydrolysis of at least 10 different concen- trations of p-nitrophenyl b-fucoside, p-nitrophenyl b-gluco- side, p-nitrophenyl b-galactoside, p-nitrophenyl b-xyloside Table 3. Parameters of the linear free energy relationships (LFER) between the wild-type and mutant Sfbgly. LFER parameter Mutant E190A E194A K201A M453A K201F E190Q Slope 1.0 0.98 1.0 1.0 0.58 0.9 Correlation coefficient 0.98 0.99 0.99 0.99 0.99 0.99 Molecular basis for specificity of a b-glycosidase L. M. F. Mendonc¸a and S. R. Marana 2544 FEBS Journal 275 (2008) 2536–2547 ª 2008 The Authors Journal compilation ª 2008 FEBS and methylumbeliferyl b-glucoside were separately measured at 30 °C. Substrate concentrations ranging from 0.2 to 4 K m were used and their hydrolysis was detected by following the production of p -nitrophenolate or methylumbeliferone. All substrates were prepared in 50 mm citrate phosphate (pH 6.0). The enzyme kinetic parameters K m and k cat were determined by fitting these data on the Michaelis–Menten equation using the enzfitter software (Elsevier-Biosoft, Cambridge, UK). The K i for linear competitive inhibitors (cellobiose, cello- triose, cellotetraose, cellopentaose, methyl b-glucoside, hexyl b-glucoside, heptyl b-glucoside, octyl b-glucoside, nonyl b-glucoside and phenyl b-glucoside) were determined by measuring the initial hydrolysis rate of at least four different concentrations of methylumbeliferyl b-glucoside in the pres- ence of at least five different concentrations of inhibitors (ranging from 0 to 4 K i ). The K i values were calculated from replots of the inhibitor concentration versus the slope of the lines observed in Lineweaver–Burk plots [29]. Calculation of thermodynamic parameters Differences in the energy of the ES à complexes (DDG à ) between a pair of different enzymes (mutant and wild-type) hydrolyzing the same substrate were calculated by the equa- tion [30,31]: DDG z ¼ÀRT lnðk cat =K m Þ mut =ðk cat =K m Þ wt ð1Þ where R is the gas constant (8.3144 JÆmol )1 ÆK )1 ), T is the absolute temperature (303 K) and mut and wt indicate mutant and wild-type Sfbgly, respectively. The binding energy between an enzyme and a competi- tive inhibitor was calculated using the equation: DG 0 ¼ÀRT lnð1=K i Þð2Þ where R is the gas constant (8.3144 JÆmol )1 ÆK )1 ) and T is the absolute temperature (303 K). The binding energy corresponding to each glucose unit of an oligocellodextrin was calculated using the equation: DDG 0 n ¼ DG 0 n À DG 0 ðnÀ1Þ ð3Þ where DDG 0 n represents the binding energy of the ‘n’ glu- cose unit of the inhibitor, DG 0 n corresponds to the binding energy of the oligocellodextrin presenting a degree of poly- merization equal to ‘n’, and DG 0 (n)1) is the binding energy of the oligocellodextrin presenting a degree of polymeriza- tion equal to ‘(n)1)’. Methyl b-glucoside was used as an inhibitor presenting a degree of polymerization equal to 1. Linear free energy relationships The effect of the alteration of the substrate structure on the stability of the complex ES à was evaluated using the follow- ing equation [30,31]: DDG z ¼ RT lnðk cat =K m Þ 1 =ðk cat =K m Þ 2 ð4Þ where R is the gas constant (8.3144 JÆmol )1 ÆK )1 ), T is the absolute temperature (303 K) and k cat ⁄ K m is the rate constant of hydrolysis of substrates 1 and 2 by the same enzyme (wild-type or mutant Sfbgly). The substrates were p-nitrophenyl b-fucoside, p-nitrophenyl b-glucoside and p-nitrophenyl b-galactoside. Then, DDG à values calculated for the wild-type Sfbgly were plotted versus those for each mutant Sfbgly. The simi- larity between the active sites of the wild-type enzyme and each mutant enzyme is related to the linear correlation coefficient and the line slope [32]. Structural comparison and sequence alignment The spatial structure of Sfbgly was modeled using chain A of the myrosinase of the aphid Brevicoryne brassicae (1WCG) as the template [33]. The homology modeling process was performed using the Swiss Model server. The resulting structure was visualized using DeepView ⁄ Swiss PDBViewer v3.7 [34]. The structure of Sfbgly, Zea mays b-glucosidase 1 (ZmGlu1; 1E56), Sorghum bicolor (SbDhr1; 1V03) and Paenibacillus polymyxa (BlgB; 2O9R) were visu- alized and superimposed using DeepView ⁄ Swiss PDBView- er v3.7. Amino acid sequences of these b-glycosidases were retrieved from the CAZy databank [2] and aligned using clustalx [35]. Only the segment containing residues form- ing the aglycone-binding site of ZmGlu1 and SbDhr1 was presented. Acknowledgements This project is supported by FAPESP (Fundac¸ a ˜ ode Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cient- ı ´ fico e Tecnolo ´ gico). L. M. F. 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