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

Abstract

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.

Figure 1

Phylogenetic tree of 52 microbial glucokinases. The phylogenetic tree shows two lineages of glucokinases, depicted as blue and red branches. Each of these lineages received high bootstrap support. Bootstrap values (500 sample runs) are expressed in percentage. GenBank accession number for each glucokinase is provided.

Figure 2

A representative of microbial glucokinases from two lineages shows the presence or absence of the CXCGX(2)GCXE motif. B. subtilis [6], B. megaterium [11], C. glutamicum [20], R. salmoninarum [16], S. coelicolor [34], B. abortus [35], E. coli [36], and Z. mobilis [37] glucokinases are followed by their GenBank accession numbers.

Figure 3

B. subtilis GlcK can form a homodimer molecule. (A) GlcK dimerization is shown on 12% SDS-PAGE under reducing condition (1) and non-reducing condition (2). Molecular mass standard 70L (Sigma, Deisenhofen, Germany) with indicated size was used. (B) Cross-link experiment of GlcK was separated on an 8% polyacrylamide gel under non-reducing condition. Non-cross linked GlcK (1) demonstrates both monomeric and homodimeric form of GlcK (*), oxidized GlcK with 1% H₂O₂ (2), and 10 mM CuSO₄.2H₂O and 30 mM 1,10-phenantroline (3) shows only the homodimer. A MALDI-TOF mass spectrum shows the homodimer (70.4 kDa) and monomer (35.2 kDa) of GlcK. The 17.6 kDa peak is the protonated form of the sample.

Figure 4

Morphology of E. coli UE26 (ptsG ptsM glk) colonies expressing B. subtilis GlcK and its mutants on 50 mM glucose MacConkey agar supplemented with 100 mM fucose.

Figure 5

Fluorescence emission spectra of B. subtilis GlcK, GlcK^C166A, GlcK^C282A, and GlcK^C321A, which were measured at excitation wavelength 280 nm (A), 295 nm (B), and subtraction of 280–295 nm. Samples (2.0 μM) were measured at 22°C in 50 mM Tris-Cl, pH 7.5. The spectra have been corrected by the buffer values. Experiments had been repeated three times.