Beta-glucoside kinase (BglK) from Klebsiella pneumoniae. Purification, properties, and preparative synthesis of 6-phospho-beta-D-glucosides

J Biol Chem. 2002 Sep 13;277(37):34310-21. doi: 10.1074/jbc.M206397200. Epub 2002 Jul 10.

Abstract

ATP-dependent beta-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC ) exists as a homotetramer composed of non-covalently linked subunits of M(r) approximately 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 (AFD(7)IG(9)GT) 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(12) approximately 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 (His6)BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200-500 mg) of ten 6-phospho-beta-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-beta-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-beta-d-glucosides have been determined by fast atom bombardment-negative ion spectrometry, thin-layer chromatography, and (1)H and (13)C 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) approximately 32,200) that exhibits approximately 58% amino acid identity with BglK. The protein encoded by the two genes exhibits beta-glucoside kinase activity and cross-reacts with polyclonal antibody to (His6)BglK from K. pneumoniae. The location of lmo2764 and lin2907 within a beta-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog in Listeria species.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Glucosides / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Klebsiella pneumoniae / enzymology*
  • Klebsiella pneumoniae / growth & development
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry
  • Phosphotransferases (Alcohol Group Acceptor) / isolation & purification*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Substrate Specificity

Substances

  • Glucosides
  • Phosphotransferases (Alcohol Group Acceptor)
  • beta-glucoside kinase

Associated data

  • GENBANK/AY035305