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

Mesak LR, Mesak FM, Dahl MK - BMC Microbiol. (2004)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nuremberg, Staudstrasse 5, 91058 Erlangen, Germany. lrmesak@yahoo.com

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.

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Fluorescence emission spectra of B. subtilis GlcK, GlcKC166A, GlcKC282A, and GlcKC321A, 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.
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Figure 5: Fluorescence emission spectra of B. subtilis GlcK, GlcKC166A, GlcKC282A, and GlcKC321A, 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.

Mentions: To prove that there were conformational changes of GlcKC282A and GlcKC321A, which had higher enzymatic activity, we analysed the GlcK mutants with fluorescence spectroscopy. Since the GlcK contains six tryptophan (W) and five tyrosine (Y) residues, excitation wavelengths of 280 and 295 nm were used to obtain emission spectra of the protein. Subtraction of W spectrum at λex.295 from the W-Y spectrum at λex.280 was done in order to obtain the separated spectrum of Y according to Isaev-Ivanov et al. [17]. GlcKC282A as well as GlcKC321A showed significant increased fluorescence intensity compared to the GlcKC166A, which has similar spectra to the wild type GlcK. This observation is indicative of changes in their structure due to the C mutation at 282 or 321. Increasing fluorescence intensities, as shown by GlcKC282A and GlcKC321A (Fig. 5), were due to conformational changes by these mutants leading to the re-positioning of tryptophan (λex.295) and tyrosine (λex.280–295) residues. Conformational changes by GlcKC321A were more pronounced as shown, not just by higher fluorescence intensity, but also a shift towards higher wavelengths (red shift) of the emission peak with respect to the wild type GlcK, GlcKC166A, and GlcKC282A (Fig. 5A, 5C). The red shift indicates a phenomenon similar to the effect of solvent reorganization. The enhanced fluorescence suggests a quenching mechanism that involves the thiol of C residue. Both red shift and enhanced fluorescence imply a loosening of packing interactions in the core of the protein and co-localization of C residues as well as W/Y residues that contribute to fluorescence [18,19]. In our case, these implications may cause an increased ability of glucokinase to phosphorylate glucose as shown by the increasing glucokinase activity of GlcKC321A (Fig. 4, 5).


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

Mesak LR, Mesak FM, Dahl MK - BMC Microbiol. (2004)

Fluorescence emission spectra of B. subtilis GlcK, GlcKC166A, GlcKC282A, and GlcKC321A, 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.
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Related In: Results  -  Collection

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Figure 5: Fluorescence emission spectra of B. subtilis GlcK, GlcKC166A, GlcKC282A, and GlcKC321A, 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.
Mentions: To prove that there were conformational changes of GlcKC282A and GlcKC321A, which had higher enzymatic activity, we analysed the GlcK mutants with fluorescence spectroscopy. Since the GlcK contains six tryptophan (W) and five tyrosine (Y) residues, excitation wavelengths of 280 and 295 nm were used to obtain emission spectra of the protein. Subtraction of W spectrum at λex.295 from the W-Y spectrum at λex.280 was done in order to obtain the separated spectrum of Y according to Isaev-Ivanov et al. [17]. GlcKC282A as well as GlcKC321A showed significant increased fluorescence intensity compared to the GlcKC166A, which has similar spectra to the wild type GlcK. This observation is indicative of changes in their structure due to the C mutation at 282 or 321. Increasing fluorescence intensities, as shown by GlcKC282A and GlcKC321A (Fig. 5), were due to conformational changes by these mutants leading to the re-positioning of tryptophan (λex.295) and tyrosine (λex.280–295) residues. Conformational changes by GlcKC321A were more pronounced as shown, not just by higher fluorescence intensity, but also a shift towards higher wavelengths (red shift) of the emission peak with respect to the wild type GlcK, GlcKC166A, and GlcKC282A (Fig. 5A, 5C). The red shift indicates a phenomenon similar to the effect of solvent reorganization. The enhanced fluorescence suggests a quenching mechanism that involves the thiol of C residue. Both red shift and enhanced fluorescence imply a loosening of packing interactions in the core of the protein and co-localization of C residues as well as W/Y residues that contribute to fluorescence [18,19]. In our case, these implications may cause an increased ability of glucokinase to phosphorylate glucose as shown by the increasing glucokinase activity of GlcKC321A (Fig. 4, 5).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nuremberg, Staudstrasse 5, 91058 Erlangen, Germany. lrmesak@yahoo.com

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.

Show MeSH
Related in: MedlinePlus