%0 Journal Article
%T Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages
%A Lili R Mesak
%A Felix M Mesak
%A Michael K Dahl
%J BMC Microbiology
%D 2004
%I BioMed Central
%R 10.1186/1471-2180-4-6
%X 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.Glucose kinase/glucokinase (GlcK/Glk) (EC 2.7.1.2) is one of the first enzymes encountered along the glycolytic pathway. This enzyme is responsible for catalyzing the ATP/ADP-dependent phosphorylation of the sixth carbon position of glucose to glucose 6-phosphate. Unlike the bacterial and archaeal glucokinases, the closest eukaryotic glucokinase counterpart such as yeast hexokinase B and human hexokinase IV (HK4 or GCK) (EC 2.7.1.1) are well characterized. In fact, the protein structure of yeast hexokinase B (31% identical amino acid residues to human HK4) was deciphered more than two decades ago [1]. Sites for the glucose formed hydrogen bond in human HK4: T168, K169, N204, D205, N231, and E290 are conserved among eukaryotes [2-4]. However, these sites are not found in microbial glucokinases. HK4 is able to phosphorylate not only glucose, but also mannose, fructose, sorbitol, and glucosamine (for review, see reference [5]). Microbial glucokinase has its own
%U http://www.biomedcentral.com/1471-2180/4/6