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PMID:9045824
| Citation |
Thompson, J, Robrish, SA, Bouma, CL, Freedberg, DI and Folk, JE (1997) Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone. J. Bacteriol. 179:1636-45 |
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| Abstract |
6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-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-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-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-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum. |
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| Keywords |
Amino Acid Sequence; Binding Sites; Cloning, Molecular; Fusobacterium/enzymology; Fusobacterium/genetics; Gluconates/metabolism; Gluconates/pharmacology; Glucosephosphate Dehydrogenase/metabolism; Glucosidases/antagonists & inhibitors; Glucosidases/genetics; Glucosidases/isolation & purification; Glucosidases/metabolism; Glucosides/metabolism; Kinetics; Lactones/metabolism; Lactones/pharmacology; Molecular Sequence Data; Molecular Weight; Sequence Alignment; Substrate Specificity |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0009313: oligosaccharide catabolic process |
ECO:0000314: |
P |
Table 2 |
complete | ||||
| GO:0008706: 6-phospho-beta-glucosidase activity |
ECO:0000314: |
F |
P-b-glucosidase activity in F. mortiferum. An extract prepared from cellobiose-grown cells of F. mortiferum rapidly hydrolyzed the chromogenic substrate pNPbGlc6P to yield Glc6P and the yellow p-nitrophenolate ion. (Rate 5 2.42 mmol of pNPbGlc6P cleaved per min per mg of protein.) The isomeric analogs pNPaGlc6P and oNP-b-galactopyranoside-6P were cleaved at 2.5% and 5.4%, respectively, of the rate of pNPbGlc6P. There was no detectable hydrolysis of pNP-agalactopyranoside or pNP-a-mannopyranoside in either phosphorylated or nonphosphorylated forms (data not shown). |
complete | ||||
See also
References
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