Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages.
BACKGROUND
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 2.7.1.2), yet glucose is merely its substrate. Our lab has previously isolated and characterized the glcK gene.


RESULTS
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.


CONCLUSIONS
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.
Mesak
Lili R
LR
Department of Microbiology, Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nuremberg, Staudstrasse 5, 91058 Erlangen, Germany. lrmesak@yahoo.com
Mesak
Felix M
FM
Dahl
Michael K
MK
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005941:Glucokinase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D005941:Glucokinase; D003545:Cysteine