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