Enzymatic mechanism of BglA-1 from Streptococcus pneumoniae TIGR4

Streptococcus pneumoniae (S. pneumoniae), the important pathogenic microorganism with high mortality rates, is one of the leading causes of acute pneumonia, otitis media, meningitis, and many other serious diseases in humans [1-3]. Pneumococcal invasions lead to more than 1.6 million infections worldwide every year. In developed and developing countries, the treatment and prevention of diseases caused by S. pneumoniae still have great burden [4,5]. S. pneumoniae can colonize on the human nasopharyngeal mucosa when the baby is born just a few months. The microorganism need abundant energy sources, such as carton sources, to satisfy its growth in host cells during infection and colonization. There are 32 carbohydrates identified as energy sources in S. pneumoniae, including three-carbon molecules (glycerol), nine hexoses or hexose derivatives, three α-galactosides, two β-galactosides, four α-glucosides, seven β-glucosides, and six polysaccharides [6]. Carbohydrates, as the important carbon source of S. pneumoniae, its accumulation is facilitated by various sugar transporters, including phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) transporters, cation/proton-coupled transporters and ATPbinding cassette (ABC) transporters [7-17]. PEP-PTS systems which is the most ubiquitous transporters in bacteria, transport various carbohydrates, such as glucose, sucrose, mannitol, fructose, lactose, mannose, and cellobiose, the β-1,4 disaccharides of D-glucose [18]. The PEP-PTS systems are composed of three components: enzyme I (EI), heat-stable phosphocarrier protein (HPr), and enzyme II (EII) [13,19]. EI and HPr are nonspecific energy-coupling components. EII is consisting of three or four functional subunits (EIIA, EIIB, EIIC, and sometimes EIID) [20]. The transmembrane permeases, EIIC and EIID, which are responsible for recognition and binding of specific sugar substrates, transport sugars across the cell membrane into the cytoplasm. The sugar molecule is subsequently transferred to EIIA and EIIB domains and phosphorylated [10]. The PEP-PTS systems transport carbohydrates coinstantaneously with phosphorylation of C6 (11). The 6-phospho-β-glucosidase BglA-1 from S. pneumoniae belongs to glycoside hydrolase family 1 (GH-1) which possesses a varity of glycosyl hydrolases. Some 6-phospho-β-glucosidases are grouped in the glycosidase families GH-4, in addition to GH-1. GH-1 family belongs to a superfamily (or clan) termed GH-A, all members of which possess a catalytic domain of (α/β)8 TIM barrel fold. Members of GH-1, such as 6-phospho-β-glucosidase (EC 3.2.1.86), 6-phosphoβ-galactosidase (EC 3.2.1.85) and β-glucosidase (EC 3.2.1.21) have different substrates specificity (see the CAZy database) [21]. The 6-phospho-β-glucosidase can hydrolyze 6-phospho-β-glucoside to produce glucose and glucose 6-phosphate (G6P), both of which can enter the energy-generating glycolytic pathway [22]. The 478-residue protein, 6-phospho-β-glucosidase BglA-1 from S. pneumoniae TIGR4 is encoded by the gene SP0303 which resides in an eight-gene operon (Figure 1A). The operon composed by a lactose-type PTS which includes three separate subunits: EIIB (SP0305), EIIA (SP0308), EIIC (SP0310) and a multidomain transcriptional regulator (Figure 1B) [23]. The primary sequence of BglA-1 is homologous to the following GH-1 enzymes of known structure: Lactococcus lactis PGALase, Bacillus polymyxa BglA, Human hCBG and S. pneumoniae BglA-2 [24-28]. The structure-based catalytic mechanism of GH-1 PGALase from Lactococcus lactis has been elucidated [24,25]. As reported previously,two conserved residues (glutamate or aspartate) function as catalytic residues, one as a nucleophile group and the other as a proton donor. The process of catalysis is as follow: the glycosidic oxygen of the substrate is protonated by the proton donor and forms the transient oxocarbenium state. Then nucleophilic residue attacks the protonated glycosidic bond, with the formation of a glycosyl-enzyme intermediate. Finally, the protonated glycosidic bond is broken by a proton provided Abstract


Experimental procedures
Cloning and expression of BglA-1 and its mutants-The coding region of the bglA-1 gene was amplified from the genomic DNA of S. pneumoniae TIGR4. The bglA-1 gene and its mutants were respectively constructed into a pET28a-derived expression vector with N-terminal hexahistidine (6×His) tags. Both the wild-type and mutants were overexpressed in Escherichia coli (E. coli) strain Rosetta (DE3) (Novagen) using 2×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 2×YT medium containing 30 μg/ml kanamycin and 34 μg/ml chloramphenicol. When the OD 600nm reached about 0.6，0.8, the recombinant proteins were induced with 0.2 mM isopropyl β-D-1thiogalactopyranoside (IPTG) for 20 h at 16°C.
Purification of BglA-1 and its mutants-The cells were collected by centrifugation at 4000×g for 15 min and resuspended in 50 ml of lysis buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl). After 5 min of sonication and 25 min of centrifugation at 12,000×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 7.5, 100 mM NaCl). The target protein was washed with the binding buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl) and eluted with the same buffer containing 250 mM imidazole. Then the target protein was loaded onto a Superdex 200 column (GE Healthcare) equilibrated with the buffer containing (20 mM Tris-Cl, pH 7.5, 100 mM NaCl). Fractions containing the target protein were combined and collected. 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 -80°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-1 as the template. The mutant proteins were expressed, purified, and stored in the same manner as the wildtype protein.
Dynamic light scattering-Dynamic light scattering was measured through a DYNAPRO-99 (Wyatt Technology Corp, 6300 Hollister) using a 532 nm green laser. The sample was measured in single-use UV-plastic cuvettes (Wyatt Technology Corp, 6300 Hollister). Firstly, a time scale of the scattered light intensity fluctuations was measured. Secondly, the instrument was equilibrated for 2 min at 25°C. At last, the molecular weight of the solution protein BglA-1 was analyzed with the software Dynamic V6 (Wyatt Technology Corp, 6300 Hollister).
Enzymatic activity assays-The enzymatic parameters of wild-type BglA-1 and its mutants were measured with the substrate p-nitrophenylβ-D-glucopyranoside-6-phosphate (pNpβGlc6P) following the previous processes with minor changes [31]. All of the reactions were performed at 37°C in the buffer containing 50 mM Na 2 HPO 4 /50 mM NaH 2 PO 4 , pH 7.5 and triggered by the addition of BglA-1. Using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA), the change in absorption at 405 nm was monitored continuously. The reaction product PNP was calculated according to a standard curve of p-nitrophenol, which was described by Prag et al. The final Michaelis-Menten parameters (V max and K m ) of BglA-1 were extracted from these data by nonlinear fitting to the Michaelis-Menten equation using the program Origin 7.5.

Preparation of 1-Phenyl-3-methyl-5-pyrazolone (PMP)
derivatives of saccharides-PMP derivatization of saccharides was implemented as described previously with minor changes [32][33][34]. Briefly, the 10 μl reaction mixture was mixed with 10 μl of 0.3 M aqueous NaOH and a 10 μl 0.5 M methanol solution of PMP. The total reaction system of 30 μl mixture was placed to react for 30 min at 70°C, After vigorous shaking and centrifuging, the organic phase in the lower was carefully abandoned to remove the excess reagents. The extraction procedure was repeated three times. At last, the aqueous phase containing derivatives was diluted with 40 μl of water before HPLC analysis.
HPLC analysis-The assays towards specific substrate were performed at 37°C in a 10 μl system containing the buffer of 50 mM Na 2 HPO 4 /50 mM NaH 2 PO 4 , pH 7.5 and the disaccharide substrate cellobiose-6p´ with kinds of concentrations. The reactions were triggered by the addition of the purified enzymes and terminated by mixing with 10 μl of 0.3 M NaOH. After PMP derivation as mentioned above, the reaction product was centrifuged at 12,000×g for 10 min, and 15 μl supernatant was analyzed by HPLC system (Agilent 1200 Series). Glucose and G6P standards were quantified by HPLC analysis using different concentrations ranging from 0.1 to 1 mM. The mixing buffer composed of 20% acetonitrile and 100 mM Na 2 HPO 4 /NaH 2 PO 4 , pH 7.0 was processed as described previously for equilibration of the column (Eclipse XDB-C18 column, 4.6×150 mm; Agilent) and separation of the components at a flow rate of 1 ml/min [34]. Retention times of monosaccharides were determined by comparison with standard monosaccharide solutions. Kinetic parameters determinations through three independent experiments were made to calculate the means and standard deviations for the K m and k cat values.
Preparation of Cellobiose-6´P, Thiocellobiose-6´P, and pNPβGlc6P-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-β-linked disaccharides was as described previously [35]. In brief, phosphorylation was effected by incubation of the disaccharides with ATP-dependent β-glucoside kinase (BglK, EC 2.7.1.85) from Klebsiella pneumoniae. Phosphorylated derivatives were first isolated by Ba 2+ 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 pNPβGlc6P was prepared by phosphorylation of the C6 hydroxyl moiety of pNP-β-Dglucopyranoside with phosphorus oxychloride in trimethyl phosphate containing a small amount of water [34].

Results and discussion
The state of BglA-1 in the solution-The plasmid of BglA-1 was constructed and overexpressed as described as the experimental procedures. The protein was purified though Ni-chelating affinity chromatography and size exclusion chromatography. And dynamic light scattering was carried out to confirm the state of BglA-1 in the solution. The results of size exclusion chromatography ( Figure  2) and dynamic light scattering confirmed the existence of BglA-1 as a dimer in solution.
The enzymatic activity of BglA-1-The samples for enzymatic activity assays were collected at the highest peak fractions of size exclusion chromatography without concentration. The purity of protein was assessed by SDS-PAGE. Firstly, we tested enzymatic activity of BglA-1 for generic substrate (pNpβGlc6P) and specific substrate (cellobiose-6´p). The K m and k cat values for generic substrate were 654±4.8 μM and 136±6.4 s -1 , respectively, and for specific substrate, 1924±8.9 μM and 152±3.8 s -1 (Table 1). These results suggested BglA-1 was a 6-phospho-β-glucosidase. Compared with other GH-1 members of known structures, the active-site residues of BglA-1 are highly conserved, especially two catalytic residues Glu 176 and Glu 375 (corresponding to Glu 171 and Glu 364 in BglA-2) (Figure 3). This suggests that BglA-1 presents a same catalytic mechanism as the previous known structures of GH-1 members [28,29]. The mutants E176A, E176Q, E375A and E375Q completely lost their activities towards pNpβGlc6P and cellobiose-6´p, suggesting the critical roles of Glu 176 and Glu 375 of BglA-1 in catalysis (Table 1).
We further tested the enzymatic activity of the wild-type and mutant proteins of BglA-1 for other substrates (gentiobiose-6´P or maltose-6´P) (Figure 4). No activity was showed towards these substrates. These results demonstrated the specificity of BglA-1 towards cellobiose-6´p.
Key residues to the substrate specificity towards +1 site of cellobiose-6p´-Sequence alignment results revealed that three residues Phe 131 , Trp 313 and Trp 349 of BglA-1 (corresponding to Tyr 126 , Trp 303 and Trp 338 in BglA-2) were generally conserved (Fig. 3). To identify the putative roles of Phe 131 , Trp 313 and Trp 349 of BglA-1, enzymatic assays towards the site-directed mutants were carried out. The mutant F131A completely lost its activity whereas F131Y retained the activity of approximately 81% compared to the wild-type ( Table 1). The results suggested that Tyr 313 is indispensable for the activity by contributing to the hydrophobic pocket. In addition, the mutant W349A also absolutely lost its activity. These results clearly demonstrated that residues Phe 131 , Tyr 313 and Trp 349 play an important role in the substrate binding and thus might determine the specificity of +1 site sugar.
Key residues to the substrate specificity towards the phosphate group of cellobiose-6´p-As described previously, the phosphatebinding residues Ser 424 , Lys 430 and Tyr 432 in BglA-2 discriminated the phosphorylated from the non-phosphorylated sugar [34]. Compared between the sequences of BglA-1 and BglA-2, the residues Ser 432 , Lys 438 and Tyr 440 of BglA-1 were highly conserved, corresponding to Ser 424 , Lys 430 and Tyr 432 of BglA-2. Sequence analysis suggested that the three residues are conserved in GH-1 6-phospho-β-glucosidases from bacteria to protozoon as described previously [28]. As demonstrated previously, the residues were substituted by non-polar residues in other GH-1 glycosidases with non-phosphorylated substrate, such as β-glucosidase A from Bacillus polymyxa [26]. To verify the roles of the three residues,   Sequence alignment indicated that two catalytic residues Glu 176 and Glu 375 , the +1 site residues, Phe 131 , Trp 313 and Trp 349 and phosphatebinding residues, Ser 432 , Lys 438 and Tyr 440 , were exclusively conserved. The results indicated that BglA-1 might adopt a similar overall structure and hydrolyze the similar substrate as other 6-phospho-β-glucosidase.
In summary, based on sequence alignment analysis and enzymatic assays, we not only elucidated the specificity towards +1 site glucose, but also the specificity towards -1 site phosphate group of 6-phosphoβ-glucosides. The results expand our understanding on the substrate recognition pattern and specificity of GH-1 enzymes.