Structural Insights into the Substrate Specificity of a 6-Phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4*

Background: Streptococcus pneumoniae BglA-2 is a GH-1 6-phospho-β-glucosidase with specificity toward 1,4-linked 6-phospho-β-glucosides. Results: BglA-2 and other GH-1 members adopt a similar overall structure and catalytic mechanism. Conclusion: Tyr126, Tyr303, and Trp338 determine substrate specificity, and Ser424, Lys430, and Tyr432 discriminate phosphorylated from non-phosphorylated substrate. A tryptophan residue discriminates 6-phospho-β-glucosidase from 6-phospho-β-galactosidase activities. Significance: BglA-2 structures provide new insight into characteristics and substrate specificity of 6-phospho-β-glucosidase. The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr126, Tyr303, and Trp338, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser424, Lys430, and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.

three-gene operon ( Fig. 2A) comprising a transcription antiterminator LicT (SP_0576) and a downstream gene BglP (SP_0577) encoding a ␤-glucoside PEP-PTS transporter. This operon is present in a number of species, including Streptococcus mutans, Clostridium longisporum, and Listeria monocytogenes. BglP comprises three domains, EIIA, EIIB, and EIIC, that facilitate the simultaneous translocation and phosphorylation of cellobiose and related ␤-glycosides (Fig. 2B). The transmembrane permease domain (EIIC) is responsible for recognition and binding of specific substrates, and the incoming sugars are phosphorylated by EIIA and EIIB domains of the PTS (17,18). BglA-2 hydrolyzes cellobiose 6Ј-phosphate (cellobiose-6ЈP) to yield G6P and glucose (Fig. 2B) that are further metabolized by the energy-generating glycolytic pathway (17,19). Based on the sequence similarity, BglA-2 is assigned to glycoside hydrolase family 1 (GH-1), which includes a variety of glycoside hydrolases, such as 6-phospho-␤-glucosidase (EC 3.  (20). Members of the GH-1 family share a common catalytic mechanism and exhibit similar structural folds, including a (␤/␣) 8 TIM-barrel. As described in Koshland's double displacement mechanisms (21,22), two conserved acidic residues (glutamate or aspartate) are catalytic residues. One of these amino acids functions as a proton donor, and the other functions as a nucleophile. First the proton donor provides a proton to the substrate to protonate the glycosidic oxygen, with the attendant formation of the transient oxocarbenium state. Then the nucleophile residue attacks the protonated glycosidic bond and forms a glycosyl-enzyme intermediate. Finally, a water molecule provides a proton to break the glycosyl-enzyme intermediate, thus restoring the enzyme to its original protonated state. Several structures of GH-1 members are now available (23)(24)(25)(26), but (to our knowledge) there are no reports of the co-crystallization of an intact substrate at the active site of any GH-1 phospho-␤-glucosidase. Significantly, the mechanism and determinants of enzyme specificity toward 1,4-linked 6-phospho-␤-glucosides remain unclear. In this work, we present the first crystal structures of BglA-2 in both the apo form and in complex with thiocellobiose-6ЈP at 2.0 and 2.4 Å, respectively. Structural analysis, in combination with the enzymatic data obtained from site-directed mutants, has enabled us to define the structural elements that contribute to enzyme recognition of 1,4-linked 6-phospho-␤-glucosides. Importantly, we provide evidence that three key residues, Ser 424 , Lys 430 , and Tyr 432 of BglA-2, play functional roles in enzyme discrimination between the hydrolysis of phosphorylated and the non-phosphorylated substrates. Finally, results obtained via site-directed mutagenesis show that a tryptophan residue plays an important role in substrate discrimination between 6-phospho-␤galactosidases and 6-phospho-␤-glucosidases in the GH-1 family.

EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of BglA-2 and Its Mutants-The coding sequence of the bglA-2 gene was amplified from the genomic DNA of S. pneumoniae TIGR4. The bglA-2 gene and its mutants were respectively cloned into the pET28a (Novagen) expression vector with an N-terminal His 6 tag. Both the wild-type and mutant proteins were overexpressed in Escherichia coli strain BL21(DE3) (Novagen) using 2ϫ YT culture medium (5 g of NaCl, 16 g of Bacto-Tryptone, and 10 g of yeast extract/liter). The transformed cells were grown at 37°C in 2ϫ YT medium containing 30 g/ml kanamycin until the A 600 nm reached 0.6 -0.8. Expression of the recombinant proteins was then induced by the addition of 0.2 mM isopropyl ␤-D-1-thiogalactopyranoside for 20 h at 16°C. The cells were collected by centrifugation at 8,000 ϫ g for 10 min and resuspended in 45 ml of lysis buffer (20 mM Tris-Cl, pH 8.0, 100 mM NaCl). After 6 min of sonication and 30 min of centrifugation at 12,000 ϫ g, the supernatant containing the target protein was collected and loaded onto a nickel-nitrilotriacetic acid column (GE Healthcare) equilibrated with the binding buffer (20 mM Tris-Cl, pH 8.0, 100 mM NaCl). The column was washed with binding buffer, and the target protein was then eluted with the same buffer containing 300 mM imidazole. The target protein was then loaded onto a Superdex 200 column  (GE Healthcare), and fractions containing the target protein were collected and pooled. The purified protein was concentrated to 10 mg/ml by ultrafiltration (Amicon, Millipore Corp.) for crystallization trials. Samples for enzymatic activity assays were collected at the highest peak fractions without concentration. The purity of protein was assessed by SDS-PAGE, and the protein sample was stored at Ϫ80°C. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the plasmid encoding the wild-type BglA-2 serving as template. The mutant proteins were expressed, purified, and stored as described above.
Crystallization, Data Collection, and Processing-Both the apo and complex forms of BglA-2 were concentrated to 10 mg/ml by ultrafiltration for crystallization. All crystals were grown at 16°C using the sitting drop vapor diffusion technique. Each drop contained 1 l of protein sample (10 mg/ml protein in buffer containing 20 mM Tris-Cl, pH 8.0, 100 mM NaCl) with an equal volume of the reservoir solution (15% polyethylene glycol 5000MME, 0.1 M sodium citrate tribasic dehydrate, pH 5.6). The crystals were transferred to cryoprotectant (reservoir solution supplemented with 25% glycerol) and flash-cooled with liquid nitrogen. All diffraction data were collected at 100 K in a liquid nitrogen stream at the Shanghai Synchrotron Radiation Facility. The data were integrated with the program Mosflm (27) and scaled with the program Scala in CCP4i (28).
Structure Determination and Refinement-The structure of apo form BglA-2 was solved by molecular replacement with MOLREP using the coordinates of 50% sequence-identical E. coli BglA (Protein Data Bank code 2XHY) as the search model. The complex form of BglA-2 (with thiocellobiose-6ЈP) was solved by molecular replacement using the apo form BglA-2 as the search model. The initial model was further refined by using the maximum likelihood method implemented in REFMAC5 (29) as part of the CCP4i (28) program suite and rebuilt interactively by using the A-weighted electron density maps with coefficients 2F o Ϫ F c and F o Ϫ F c in the program COOT (30). The final model was evaluated with the programs MOLPROBITY (31) and PROCHECK (32). The data collection and structure refinement statistics of apo form and complex form BglA-2 are listed in Table 1. All of the structure figures were prepared with the program PyMOL (33).
Enzymatic Activity Assays-The kinetic parameters of wild-type BglA-2 and its mutants were determined using chromogenic p-nitrophenyl-␤-D-glucopyranoside 6-phosphate (pNP␤Glc6P) as substrate (34). All assays were performed at 37°C in a buffer containing 50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , pH 7.5, and reactions were initiated by the addition of BglA-2. Changes in absorption at 405 nm (formation of p-nitrophenol) were monitored continuously using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA). The reaction product p-nitrophenol was calculated from a standard curve of p-nitrophenol, as described by Prag et al. (35). Michaelis-Menten parameters (V max and K m ) of BglA-2 were extracted from these data by nonlinear fitting to the Michaelis-Menten equation using the program Origin version 7.5.

Preparation of 1-Phenyl-3-methyl-5-pyrazolone (PMP)
Derivatives-PMP derivation of saccharides was performed as described previously (36 -38) with minor changes. Briefly, 10 l of reaction mixture was mixed with 10 l of 0.3 M aqueous NaOH and 10 l of 0.5 M methanol solution of PMP. The total reaction mixture (30 l) was maintained at 70°C for 30 min, cooled to room temperature, and neutralized by the addition of 10 l of 0.3 M HCl. The solution was further mixed with 100 l of chloroform. After vigorous shaking and centrifugation, the organic phase was carefully removed to eliminate excess reagents. The extraction procedure was repeated three times. Finally, the aqueous phase containing derivatives was diluted with 40 l of water prior to HPLC analysis.
HPLC Analysis-The assays with specific substrate were performed at 37°C in a 10-l system containing the buffer of 50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , pH 7.5, and the disaccharide substrate (e.g. cellobiose-6ЈP) at a range of concentrations. The reactions were initiated by the addition of the purified enzymes and were terminated by the addition of 10 l of 0.3 M NaOH. After PMP derivation, the reaction product was centrifuged at 12,000 ϫ g for 10 min, and 15 l of supernatant was analyzed by an HPLC system (Agilent 1200 series). Glucose and G6P standards were quantified by HPLC analysis using various concentrations ranging from 0.1 to 1 mM. The mixing buffer containing 20% acetonitrile and 100 mM Na 2 HPO 4 /NaH 2 PO 4 , pH 7.0 was processed as described previously (38) for equilibration of the column (Eclipse XDB-C18 column, 4.6 ϫ 150 mm; Agilent), and separation of the components was effected at a flow rate of 1 ml/min. Retention times of monosaccharides were determined by comparison with standard solutions. Kinetic parameters were derived from three inde- is the intensity of an observation, and ͗I(hkl)͘ is the mean value for its unique reflection. Summations are over all reflections.
where F o and F c are the observed and calculated structure factor amplitudes, respectively. d R-free was calculated with 5% of the data excluded from the refinement. e Root mean square deviation from ideal values. f Categories were defined by Molprobity. pendent experiments in order to calculate the means and S.D. for the K m and k cat values.
Preparation of Cellobiose-6ЈP, Thiocellobiose-6ЈP, and pNP␤Glc6P-Cellobiose was obtained from Pfanstiehl Laboratories, and thiocellobiose was purchased from Toronto Research Chemicals. Phosphorylation of the primary hydroxyl groups of the non-reducing glucose moiety in these O-␤-linked disaccharides was as described previously (39). In brief, phosphorylation was effected by incubation of the disaccharides with ATP-dependent ␤-glucoside kinase (BglK, EC 2.7.1.85) from Klebsiella pneumoniae. Phosphorylated derivatives were first isolated by Ba 2ϩ and ethanol precipitation and further purified by ion exchange and paper chromatography. Structures and product purity were confirmed by thin layer chromatography, mass spectrometry, and NMR spectroscopy. Chromogenic pNP␤Glc6P was prepared by phosphorylation of the C6 hydroxyl moiety of pNP-␤-D-glucopyranoside with phosphorus oxychloride in trimethyl phosphate containing a small amount of water (34). Lactose-6Ј-phosphate (lactose-6ЈP) is not commercially available, and the chromogenic analog o-nitro-phenyl-␤-D-galactopyranoside 6-phosphate (oNP␤Gal6P; Sigma-Aldrich) was used as a substitute substrate for kinetics analyses.

RESULTS AND DISCUSSION
Overall Structure-The crystal structure of apo form BglA-2 was determined at 2.0 Å resolution in the space group C2. Each asymmetric unit contains two molecules, which form a stable dimer with a buried interface area of ϳ3,300 Å 2 (Fig. 3A). The dimerization in the crystal structure is consistent with the results obtained from size exclusion chromatography (supplemental Fig. S1). The dimer interface, composed of four ␣-helices (␣8, ␣9, ␣11, and ␣12) and three loops from one subunit, is stabilized primarily by a number of polar residues via hydrogen bond networks and salt bridge interactions. Similar to other reported GH-1 members, BglA-2 adopts a typical (␤/␣) 8 TIMbarrel: a central eight-stranded (␤1-␤5, ␤7, ␤9, and ␤12) parallel ␤-sheet surrounded by eight helices (␣2, ␣3, ␣5, ␣7, ␣8, and ␣11-13). In addition, the central TIM-barrel is packed by four additional helices (␣1, ␣6, ␣9, and ␣10) and six ␤-strands To date, the crystal structures of four 6-phospho-␤-glycosidases from the GH-1 family have been solved, including three 6-phospho-␤-glucosidases (E. coli BglA, S. mutans putative 6-phospho-␤-glucosidase Bgl, and Lactobacillus plantarum Pbg1) and one 6-phospho-␤-galactosidase (PGALase) from Lactococcus lactis. These proteins assume a structure similar to that of BglA-2 with an overall root mean square deviation of 2.0, 1.2, 1.3, and 1.4 Å, respectively. The main differences reside in the architecture surrounding the active site pockets. In the complex structure of PGALase with galactose 6-phosphate (galactose-6ЈP), three antiparallel ␤-strands and an 11-residue loop (Gln 305 -Arg 334 ) cover the catalytic pocket, whereas in BglA-2, the corresponding region (Asn 316 -Gly 324 ) is absent from the structure. In BglA, the loop (Asp 38 -Pro 62 ) caps the entrance of the substrate tunnel, whereas in BglA-2, the corresponding loop (Leu 36 -Leu 52 ) is considerably shorter. Despite a similar catalytic mechanism, the variable loops around the active sites may reflect the substrate specificity of each enzyme, which is in agreement with the variety of substrates hydrolyzed by GH-1 members.
The Active Site-As is evident from the BglA-2 complex structure, thiocellobiose-6ЈP is stabilized at the active site pocket via hydrophilic and hydrophobic interactions (Fig. 3B). In detail, the phosphate group that projects toward the base of the pocket is hydrogen-bonded by the side chains of Ser 424 , Lys 430 , and Tyr 432 . In subsite Ϫ1 G6P is anchored by hydrophobic interaction with Trp 415 and is also hydrogen-bonded to one of the catalytic residues (Glu 364 ) and a water molecule, Wat-1. The side chain of the catalytic residue Glu 171 resides ϳ6 Å from the glycosidic bond. Subsite ϩ1 is occupied by glucose and includes three aromatic residues: Tyr 126 , Tyr 303 , and Trp 338 . In addition, Tyr 303 makes a hydrogen bond with the carboxyl group of Glu 364 , presumably to orient and maintain Glu 364 in a conformation favorable for catalysis.
When compared with the representative complex structure of PGALase with galactose-6ЈP in the GH-1 family, many of the active site residues of BglA-2 can be superimposed, especially the two catalytic residues Glu 364 and Glu 171 (corresponding to Glu 375 and Glu 160 of PGALase and Glu 375 and Glu 176 of Bgl) (23). Notably, the distances between the side chains of PGALase Glu 160 and Bgl Glu 176 and the glycosidic bond are 3.4 and 2.8 Å, respectively, which are favorable for catalysis. Structural comparison of BglA-2 with PGALase and Bgl reveals that the thiocellobiose-6ЈP adopts a similar position but different conformation. Thus, our structure might be a complex for the interactions of thiocellobiose-6ЈP with BglA-2 that is not poised for catalysis.
Key Residues Contributing to Specificity at Subsite ϩ1-GH-1 enzymes with known structures are reported to specifically catalyze the hydrolysis of 1,4-linked non-phosphorylated ␤-glycosides or 1,4-linked 6-phospho-␤-glycosides. ␤-Glucosidase A from Bacillus polymyxa reportedly hydrolyzes ␤-1,4linked oligosaccharides composed of more than 2 units of glucose (25), and human cytosolic ␤-glucosidase hydrolyzes certain flavonoid glucosides (26). The enzyme PGALase from L. lactis was reported to hydrolyze lactose-6ЈP, formed during transport and phosphorylation via the lactose PEP-PTS (23). The model of lactose-6ЈP bound to PGALase predicted that Trp 347 at the channel entrance and Tyr 299 and Trp 421 at the substrate cavity were involved in guiding and binding the substrate by hydrophobic interactions (24). Compared with BglA-2, Trp 421 of PGALase corresponds to subsite Ϫ1 Trp 415 , whereas Tyr 299 and Trp 347 of PGALase are aligned with subsite ϩ1 Tyr 303 and Trp 338 , respectively. Although the structures of ␤-glucosidase A, cytosolic ␤-glucosidase, and PGALase are known, a lack of enzymatic assays and 6-phospho-␤glucoside complexed structure leaves the substrate specificity of 6-phospho-␤-glucosidases still ambiguous. To address the structural basis of the substrate specificity toward 1,4-linked 6-phospho-␤-glucosides, concerted efforts were made to obtain crystals of  ND  ND  ND  ND  ND  E171Q  ND  ND  ND  ND  ND  ND  E364A  ND  ND  ND  ND  ND  ND  E364Q  ND  ND  ND  ND  ND  ND  Y126A  ND  ND  ND  ND  ND  ND  Y126F 598 BglA-2 in complex with cellobiose-6ЈP, pNP␤Glc6P, glucose, G6P, and thiocellobiose-6ЈP. Fortuitously, phospho-␤-glucosidases in family GH-1 are unable to hydrolyze this phosphorylated cellobiose analog. (By contrast, phospho-␤-glucosidases assigned to family GH-4 readily cleave thiocellobiose-6ЈP by a catalytically unique series of oxidation-elimination-addition and reduction reactions (40,41).) After numerous attempts, the structure of BglA-2 in complex with thiocellobiose-6ЈP was successfully solved. Inspection of the complex shows that the hexose ring at the subsite ϩ1 moiety is orientated almost perpendicular to that of the G6P in subsite Ϫ1. The glucose moiety at subsite ϩ1 is sandwiched by residues Tyr 303 and Trp 338 on one side and on the opposite side by Tyr 126 (Fig. 3B). Among the three subsite ϩ1 residues, Tyr 126 is newly identified in our structure and corresponds to Phe 137 in PGALase. Multiple-sequence alignment among 6-phospho-␤-glycosidases in the GH-1 family shows that Tyr 126 , Tyr 303 , and Trp 338 are generally conserved in bacteria, besides the only protozoa, Leishmania infantum (Fig. 4). To clarify the roles of Tyr 126 , Tyr 303 , and Trp 338 in BglA-2 catalysis, we first determined the equilibrium dissociation constants (K d ) of both wild-type and mutant proteins of BglA-2 (Y126A, Y303A, Y303F, and W338A) toward cellobiose-6ЈP by fluorescence spectrometry. The K d values of mutants increased ϳ6 -7-fold compared with the wild type (supplemental Table S1), suggesting that the mutants had much lower cellobiose-6ЈP binding affinities compared with the wild type. Subsequently, we compared the enzymatic activities of these mutants with that of the wild type. The mutant Y126A was devoid of all activity, whereas Y126F retained ϳ59% of that of the wild type, indicative of the important role of Tyr 126 in this hydrophobic pocket. Neither the mutant Y303A nor Y303F showed any enzymatic activity (Table 2). These results show that Tyr 303 is essential for activity, not only by contributing to the hydrophobicity of the pocket but also by stabilizing the orientation of the catalytically functional carboxyl group of Glu 364 . This result is in accordance with the complex structure, in which Tyr 303 makes a hydrogen bond with the carboxyl group of Glu 364 (Fig. 3B). The mutant W338A was also completely inactive.
To further study the subsite ϩ1 specificity, we investigated the activity of the wild type and mutants of BglA-2 toward two stereoisomers of cellobiose-6ЈP, namely gentiobiose-6ЈP (␤-1,6-linked glucose 6-phosphate and glucose) and maltose-6ЈP (␣-1,4-linked glucose 6-phosphate and glucose). Neither wild-type nor mutant proteins hydrolyzed gentiobiose-6ЈP or maltose-6ЈP (supplemental Fig. S2). These results established the specificity of BglA-2 toward cellobiose-6ЈP. Furthermore, it is evident that the active site residues Tyr 126 , Tyr 303 , and Trp 338 play important roles not only in substrate binding but also in recognition of the unique spatial orientation of glucose (in cellobiose-6ЈP) that permits this moiety to occupy subsite ϩ1 of BglA-2.
Key Residues That Dictate Specificity toward the Phosphate Group of Cellobiose-6ЈP-As described previously, the phosphate-binding residues Ser 428 , Lys 435 , and Tyr 437 in PGALase discriminate the phosphorylated from the non-phosphorylated sugar (24). In BglA-2, three residues (Ser 424 , Lys 430 , and Tyr 432 ) make hydrogen bonds with the phosphate group of thiocello-biose-6ЈP (Fig. 3B). Comparison of the complex structures of BglA-2 and PGALase shows that residues Lys 430 and Tyr 432 of BglA-2 adopt a position similar to Lys 435 and Tyr 437 of PGALase, respectively. However, Ser 424 of BglA-2 corresponds to Ser 430 rather than Ser 428 of PGALase. Sequence analysis suggested that Lys 430 and Tyr 432 of BglA-2 are conserved in GH-1 6-phospho-␤-glycosidases (Fig. 4). However, they are substituted by non-polar residues in other GH-1 glycosidases hydrolyzing non-phosphorylated substrates, such as ␤-glucosidase A from B. polymyxa (25). Indeed, BglA-2 shows no hydrolytic activity toward non-phosphorylated cellobiose. To verify the roles of the three residues, we measured the enzymatic activities toward cellobiose-6ЈP through use of several mutants. The results showed that mutations S424A, K430A, and Y432F abolished all activity, thereby confirming the key roles of Ser 424 , Lys 430 , and Tyr 432 of BglA-2 in determining specificity toward phosphorylated substrate ( Table 2).
Sequence alignment of 6-phospho-␤-glycosidases in the GH-1 family indicates that two catalytic residues (Glu 364 and Glu 171 ); the subsite ϩ1 residues Tyr 126 , Tyr 303 , and Trp 338 ; and the phosphate-binding residues Lys 430 and Tyr 432 are exclusively conserved. The observations suggest that these homologs might adopt a similar overall structure and hydrolyze phosphorylated substrates (Fig. 4). Our structural analyses provide new insight into the substrate binding patterns and determinants of specificity of GH-1 family phospho-␤-glycosidases.
A Tryptophan Residue Discriminates 6-Phospho-␤-galactosidase from 6-Phospho-␤-glucosidase in GH-1 Family-In GH-1 family, two types of enzymes can hydrolyze phosphorylated substrates. 6-Phospho-␤-glucosidase and 6-phospho-␤galactosidase are distinguished by their subsite Ϫ1 sugars, with G6P for 6-phospho-␤-glucosidase and galactose-6ЈP for 6-phospho-␤-galactosidase. Whereas BglA-2 hydrolyzes cellobiose-6ЈP, PGALase hydrolyzes lactose-6ЈP. The major difference between cellobiose-6ЈP and lactose-6ЈP resides in the orientation of the C4 hydroxyl group of the hexose-6P compounds at subsite Ϫ1. In the case of cellobiose-6ЈP, the equatorial C4 hydroxyl lies on the opposite side of the C1 hydroxyl, whereas in lactose-6ЈP, the axial C4 hydroxyl moiety lies on the same side as the C1 hydroxyl group. In BglA-2, the subsite Ϫ1 sugar (G6P) shifts 2.4 Å apart and rotates about 67°from that of Gal6P in PGALase. This difference between the substrates is in accordance with the active site conformations. In PGALase, the C4 hydroxyl moiety is hydrogen-bonded by Trp 429 (24), which is substituted by Met 423 in BglA-2 or Ala 431 in Bgl (Fig. 5, A and  B). However, Met 423 in BglA-2 and Ala 431 in Bgl have no interaction with the subsite Ϫ1 sugar. Indeed, no residue was found to stabilize the C4 hydroxyl group of BglA-2 and Bgl. The M423A and M423W mutants of BglA-2 retain about 80 and 67% of the activity of the wild-type enzyme respectively, suggesting a non-essential role for Met 423 in catalysis (Table 2 and  supplemental Table S2). In further studies, the activities of the wild-type BglA-2 and M423W mutant were determined using oNP␤Gal6P as a chromogenic substitute for lactose-6ЈP. As expected, the wild-type BglA-2 has no detectable activity toward oNP␤Gal6P. Remarkably, the single mutation M423W elicited hydrolytic activity toward oNP␤Gal6P with a k cat /K m value of 8.6 Ϯ 2.1 ϫ 10 Ϫ5 s Ϫ1 M Ϫ1 (supplemental Table S2).
These results show that a tryptophan residue of PGALase determines that subsite Ϫ1 of this 6-phospho-␤-galactosidase is occupied by galactose-6ЈP.
Sequence analysis revealed an ϳ50% sequence homology between these two types of enzymes, suggesting a similar structural fold of a (␤/␣) 8 TIM-barrel and a common catalytic mechanism (Fig. 4). However, Trp 429 of PGALase is exclusively conserved in 6-phospho-␤-galactosidases but is usually alanine in 6-phospho-␤-glucosidases (Fig. 4). We hypothesize that both enzyme species evolved from a common ancestor, but at some point 6-phospho-␤-galactosidases evolved independently, such that a tryptophan residue was acquired in order to accommodate galactose-6ЈP (rather than G6P) at the active site of subsite Ϫ1.
6-Phospho-␤-glycosidases Provide Alternative (but Non-essential) Pathways for the Utilization of ␤-Linked Disaccharides-Multiple-sequence alignment among 6-phospho-␤-glycosidases in GH-1 family shows that they are generally conserved in bacteria, with only the protozoa L. infantum as the exception (Fig. 4). Abundant bacterial growth is dependent upon the uptake of various carbohydrates from the environment. As noted previously, there are several pathways for the uptake of carbohydrates in bacterial species, including PEP-PTS transporters (7)(8)(9)(10)(11), cation/proton-coupled transporters (12,13), and ATP-binding cassette transporters (14 -16). Among these, the group translocation PEP-PTS system is perhaps the most ubiquitous. Many species possess operons that encode genes for a glycosidase and the appropriate PEP-PTS (4). The PEP-PTS system enables organisms such as S. pneumoniae to obtain their metabolic energy via the simultaneous transport and phosphorylation of disaccharides. Intracellular 6-phospho-␤glycosidases hydrolyze these phosphorylated compounds to metabolizable monosaccharides that furnish the requisite energy for growth of microorganisms. With the exception of L. infantum, 6-phospho-␤-glycosidases exist only in bacteria, and it is likely that BglA-2 and other 6-phospho-␤-glycosidases in the GH-1 family have evolved in bacteria simultaneously with the PEP-PTS to permit the dissimilation of environmental disaccharides. The exceptional case of L. infantum may be the result of gene transfer from bacteria, as reported previously (42).
Inspection of the 2.16-megabase genome of S. pneumoniae TIGR4 reveals three PEP-PTS systems (SP_0303-10, SP_0577, and SP_2021-4), and a sugar efflux transporter that may participate in the utilization and metabolism of cellobiose by this organism (4,5,43). For the PEP-PTS systems, cellobiose should be phosphorylated to cellobiose-6ЈP prior to hydrolysis by 6-phospho-␤-glucosidases, such as BglA-2 of the SP_0577 PTS operon and SP_0303 of the SP_0303-10 PTS operon. Alternatively, cellobiose may be transported into the cell via a sugar efflux transporter and hydrolyzed intracellularly by putative cellobiase(s) encoded by SP_2021 and SP_0265. As reported previously (4), growth of S. pneumoniae on cellobiose is essentially abolished upon deletion of the transporter gene SP_0310 of the SP_0305-10 PTS, suggesting that this PEP-PTS may be essential for the uptake of cellobiose. By contrast, the ⌬SP_0577 strain shows a doubled generation time (195 min) and a prolonged lag period compared with the wild-type strain on methyl-␤-glucoside, suggesting that SP_0577 PTS is responsible for the uptake of this and related ␤-glucosides (4,18).
Each of the three PEP-PTS systems has a cellobiose/cellobiose-6ЈP hydrolase that is encoded by the SP_0303, BglA-2, and SP_2021 gene, respectively. Structure-based sequence alignment confirms that SP_0303 and BglA-2 are 6-phospho-␤-glucosidases, whereas SP_2021 is most likely a cellobiase. To test whether SP_0303 and/or BglA-2 are essential for the utilization of cellobiose, we constructed ⌬BglA-2 and ⌬BglA-2&⌬SP_0303 strains and recorded the growth profiles. The results showed no significant difference of growth rate on cellobiose between the wild-type and deletion strains (supplemental Fig. S3). Although a functional 6-phospho-␤-glucosidase may not be essential for the utilization of cellobiose, 6-phospho-␤-glycosidases encoded by PEP-PTS systems provide S. pneumoniae with alternate pathways for dissimilation of this carbohydrate and other ␤-linked disaccharides.
In summary, we have solved the crystal structure of BglA-2 in complex with the non-metabolizable analog, thiocellobiose-6ЈP. Based on structural analysis and enzymatic assays, we have clarified issues pertaining to the architectural environments of subsites Ϫ1 and ϩ1 and have identified residues that interact FIGURE 4. Multiple-sequence alignment of BglA-2 and homologs. The 6-phospho-␤-glucosidases and 6-phospho-␤-galactosidases are labeled with red and black titles, respectively. Two conserved catalytic glutamate residues and the subsite Ϫ1 residue Trp 415 are depicted by green triangles. The subsite ϩ1 residues and the phosphate-binding residues are marked with red and black triangles, respectively. The tryptophan residue discriminating 6-phospho-␤-galactosidase from 6-phospho-␤-glucosidase in GH-1 family is indicated by a blue triangle. with the phosphate moiety of G6P at subsite Ϫ1 of 6-phospho-␤-glucosidase. Importantly, we present evidence that a tryptophan residue plays a functional role in the differentiation of the catalytic properties of 6-phospho-␤-galactosidases and those of 6-phospho-␤-glucosidases assigned to GH-1 of the glycoside hydrolase family.