Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone

Abstract

6-Phosphoryl-␤-D-glucopyranosyl:6-phosphoglucohydrolase (P-␤-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-␤-glucosidase (M r , 53,000; pI, 4.5) was induced by growth of F. mortiferum on ␤-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-␤-D-glucopyranoside-6-phosphate (pNP␤Glc6P) and 4-methylumbelliferyl-␤-D-glucopyranoside-6-phosphate (4MU␤Glc6P), respectively, were used for the assay of P-␤-glucosidase activity. The enzyme hydrolyzed several P-␤-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-␤-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-␦-lactone (P-glucono-␦-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-␤-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-␤-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-␦-lactone to the enzyme from F. mortiferum.Fusobacteria are important human pathogens that collectively comprise a genus of the Bacteroidaceae family of microorganisms (14, 23). Most species of Fusobacteria rely upon amino acid fermentation to provide requisite energy for growth, but F. mortiferum has the additional capacity to utilize a wide variety of carbohydrates, including monosaccharides and ␣-and ␤-glucosides as fermentable energy sources. Our interest in the mechanisms of amino acid and sugar utilization by Fusobacteria stems from the fact that the end products of these metabolic pathways include lactic, acetic, propionic, and butyric acids. These organic acids are cytotoxic for epithelial and other tissue cells, and Fusobacteria are believed to be causative agents or a contributing factor in the etiology of oral and other diseases.Studies in our laboratory (30,31,45) provided the first evidence for the operation of the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP:PTS) in Fusobacteria. Although discovered serendipitously by Saul Roseman and his colleagues in Escherichia coli (16,34), this multicomponent system (35) is now recognized as the primary mechanism for the simultaneous translocation and phosphorylation of sugars by bacteria from both gram-positive (11, 29, 43) and gram-negative (22, 28) genera. Catabolism of disaccharides that are accumulated by the PEP-PTS as phosphorylated derivatives requires the cleavage of these compounds by intracellular substrate-sp...

Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis

“…1). Belaich et al (6) (34). Family 30 CBMs appear to be closely related to a family 9 catalytic module, although the first example, F. succinogenes CelF, coexists with the family 51 catalytic module.…”

Listeria monocytogenes 6-Phosphogluconolactonase Mutants Induce Increased Activation of a Host Cytosolic Surveillance Pathway

“…After growth in rich medium, microarray analysis revealed that pgl mutant L. monocytogenes had increased expression of several ␤-glucosidases compared to the wt. We speculate that this is due to the known inhibition of ␤-glucosidases by 6-phosphogluconolactone (36), the substrate of Pgl that is predicted to accumulate in its absence. In E. coli lacking pgl, 6-phosphogluconolactone is dephosphorylated prior to its export and hydrolysis into gluconate (14).…”

“…Therefore, Pgl, though likely important for the optimal efficiency of the PPP, may not be essential (13,14). In Escherichia coli, disruption of pgl (ybhE) results in accumulation of the Pgl substrate, 6-phosphogluconolactone (14), a reactive electrophilic molecule (20,27) and a potent inhibitor of bacterial ␤-glucosidases (36). In an E. coli pgl deletion mutant, the accumulating 6-phosphogluconolactone is dephosphorylated and exported, where it subsequently hydrolyzes to form gluconate (14).…”

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

“…Preparation of NP G6P was by the procedure described earlier for the synthesis of NP G6P. 17,28,29) Expression and purification of the enzyme. E. coli strain BL21 (DE3) carrying pET-21a(+)/BglA was grown at 37 C to mid-log phase in LB medium containing 50 mg/ml ampicillin.…”