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Journal of Bacteriology, December 1999, p. 7339-7345, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cellobiose-6-Phosphate Hydrolase (CelF) of Escherichia coli: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases

John Thompson,1,* Sergei B. Ruvinov,2 Darón I. Freedberg,3 and Barry G. Hall4

Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research,1 and Laboratory of Biochemistry, National Heart, Lung, and Blood Institute,2 National Institutes of Health, and Laboratory of Biophysics, Center for Biologics Evaluation and Research, Food and Drug Administration,3 Bethesda, Maryland 20892, and Biology Department, University of Rochester, Rochester, New York 14627-02114

Received 21 July 1999/Accepted 22 September 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 (Mw, ~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 (Mr, ~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; Mr = 50,512). Comparative sequence alignments provide tentative identification of the NAD+-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu112 and Glu356) of CelF.

* Corresponding author. Mailing address: National Institutes of Health, Bldg. 30, Room 528, Convent Dr. 4350, Bethesda, MD 20892-4350. Phone: (301) 496-4083. Fax: (301) 402-0396. E-mail: jthompson{at}dir.nidcr.nih.gov.

Journal of Bacteriology, December 1999, p. 7339-7345, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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