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Open Access Research article

Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages

Lili R Mesak12, Felix M Mesak3* and Michael K Dahl1

Author Affiliations

1 Department of Microbiology, Institute for Microbiology, Biochemistry and Genetics University of Erlangen-Nuremberg, Staudstrasse 5, 91058 Erlangen, and Department of Biology, University of Konstanz, Universitaetstrasse 1, 78457 Konstanz, Germany

2 Current address: 412-2870 Cedarwood Dr., Ottawa, ON, K1V 8Y5, Canada

3 Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, and Faculty of Medicine, University of Ottawa, 503 Smyth Rd., Ottawa, ON, K1H 1C4, Canada

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BMC Microbiology 2004, 4:6  doi:10.1186/1471-2180-4-6

Published: 3 February 2004



Bacillus subtilis glucokinase (GlcK) (GenBank NP_390365) is an ATP-dependent kinase that phosphorylates glucose to glucose 6-phosphate. The GlcK protein has very low sequence identity (13.7%) to the Escherichia coli glucokinase (Glk) (GenBank P46880) and some other glucokinases (EC, yet glucose is merely its substrate. Our lab has previously isolated and characterized the glcK gene.


Microbial glucokinases can be grouped into two different lineages. One of the lineages contains three conserved cysteine (C) residues in a CXCGX(2)GCXE motif. This motif is also present in the B. subtilis GlcK. The GlcK protein occurs in both monomer and homodimer. Each GlcK monomer has six cysteines. All cysteine residues have been mutated, one-by-one, into alanine (A). The in vivo GlcK enzymatic activity was assayed by functional complementation in E. coli UE26 (ptsG ptsM glk). Mutation of the three motif-specific residues led to an inactive enzyme. The other mutated forms retained, or in one case (GlcKC321A) even gained, activity. The fluorescence spectra of the GlcKC321A showed a red shift and enhanced fluorescence intensity compare to the wild type's.


Our results emphasize the necessity of cysteines within the CXCGX(2)GCXE motif for GlcK activity. On the other hand, the C321A mutation led to higher GlcKC321A enzymatic activity with respect to the wild type's, suggesting more adequate glucose phosphorylation.