Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone.
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.
Thompson
J
J
Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA. jthompson@yoda.nidr.nih.gov
Robrish
S A
SA
Bouma
C L
CL
Freedberg
D I
DI
Folk
J E
JE
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005673:Fusobacterium / Q000201:enzymology* / Q000235:genetics; D005942:Gluconates / Q000378:metabolism / Q000494:pharmacology*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013379:Substrate Specificity
D005942:Gluconates; D005960:Glucosides; D007783:Lactones; C048956:6-phosphonoglucono-delta-lactone; D005954:Glucosephosphate Dehydrogenase; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase