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J. Biol. Chem., Vol. 277, Issue 37, 34310-34321, September 13, 2002

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-Glucoside Kinase (BglK) from Klebsiella pneumoniae
PURIFICATION, PROPERTIES, AND PREPARATIVE SYNTHESIS OF 6-PHOSPHO--D-GLUCOSIDES^*

John Thompson^§, Frieder W. Lichtenthaler^¶, Siegfried Peters^¶, and Andreas Pikis

From the  Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892 and the ^¶ Institut für Organische Chemie, Technische Universität Darmstadt, Darmstadt D-64287, Germany

ATP-dependent -glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC 2.7.1.85) exists as a homotetramer composed of non-covalently linked subunits of M_r ~33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA of K. pneumoniae. The open reading frame (ORF) of bglK encodes a 297-residue polypeptide of calculated M_r 32,697. A motif of 7 amino acids (AFD⁷IG⁹GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t_1/2 ~ 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF, Kinase) family of proteins. Preparation of ^His6BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200-500 mg) of ten 6-phospho--D-glucosides, including cellobiose-6'-P, gentiobiose-6'-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho--glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho--D-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and ¹H and ¹³C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (M_r ~ 32,200) that exhibits ~58% amino acid identity with BglK. The protein encoded by the two genes exhibits -glucoside kinase activity and cross-reacts with polyclonal antibody to ^His6BglK from K. pneumoniae. The location of lmo2764 and lin2907 within a -glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog in Listeria species.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY035305.

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