Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases.
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.
Thompson
J
J
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, Bethesda, Maryland 20892, USA. jthompson@dir.nidcr.nih.gov
Ruvinov
S B
SB
Freedberg
D I
DI
Hall
B G
BG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005959:Glucosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005959:Glucosidases; D000520:alpha-Glucosidases; C066586:6-phospho-beta-glucosidase