PMID:10572139
| Citation |
Thompson, J, Ruvinov, SB, Freedberg, DI and Hall, BG (1999) Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases. J. Bacteriol. 181:7339-45 |
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| Abstract |
The gene celF of the cryptic cel operon of Escherichia coli has been cloned, and the encoded 6-phospho-beta-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86]) has been expressed and purified in a catalytically active state. Among phospho-beta-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD(+) for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-beta-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-beta-glucoside-6P, and the chromogenic analog, p-nitrophenyl-beta-D-glucopyranoside-6P. In the absence of a metal ion and NAD(+), purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD(+) appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (M(w), approximately 200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (M(r), approximately 50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues; M(r) = 50,512). Comparative sequence alignments provide tentative identification of the NAD(+)-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu(112) and Glu(356)) of CelF. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Catalysis; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Glucosidases/genetics; Glucosidases/isolation & purification; Glucosidases/metabolism; Kinetics; Molecular Sequence Data; Multigene Family; Phylogeny; Sequence Homology, Amino Acid; alpha-Glucosidases/genetics; alpha-Glucosidases/metabolism |
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