Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases

• Published in: Journal of bacteriology 1999-12, Vol.181 (23), p.7339-7345
• Main Authors: Thompson, J, Ruvinov, S B, Freedberg, D I, Hall, B G
• Format: Article
• Language: English
• Subjects: alpha-Glucosidases - genetics; alpha-Glucosidases - metabolism; Amino Acid Sequence; Amino acids; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Catalysis; Cloning; Electrophoresis, Polyacrylamide Gel; Enzymes and Proteins; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Genes; Glucosidases - genetics; Glucosidases - isolation & purification; Glucosidases - metabolism; Kinetics; Molecular Sequence Data; Multigene Family; Phylogeny; Sequence Homology, Amino Acid
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.181.23.7339-7345.1999

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.