Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages
© Mesak et al; licensee BioMed Central Ltd. 2004
Received: 16 October 2003
Accepted: 03 February 2004
Published: 03 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 220.127.116.11), 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.
Glucose kinase/glucokinase (GlcK/Glk) (EC 18.104.22.168) 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 22.214.171.124) 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 . 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 ). Microbial glucokinase has its own unique glucose-binding domain, which maybe conserved among glucokinases. The domain seems highly specific for glucose.
We have previously cloned and characterized the glcK gene of Bacillus subtilis . Replacement of ATP by ADP revealed no detectable glucokinase activity, neither did replacement of glucose by fructose, galactose, or mannose . The GlcK protein was characterized by K m values for ATP and glucose of 0.77 mM and 0.24 mM, respectively . The ATP binding-motif D [ILV]G [GA] [T] conserved for both GlcK and HK4 are located at the N-terminal . The mechanism of Mg2+-ATP binding to GlcK has never been directly observed but rather proposed by their homology to the ATP-binding sites of HK4 . Two recent 3-D protein structures of ADP-dependent glucokinases, belonging to hyperthermophilic archaeon, Thermococcus litoralis and Pyrococcus horikoshii, showed the Mg2+ motif (NEXE) and the ADP/ATP-dependent kinases motif ([SD]TXG XGDX [IF]) [7, 8]. Interestingly, the T. litoralis and P. horikoshii glucokinases are similar to ATP-dependent kinases: E. coli ribokinase and human adenosine kinase . As a consequence, those archaeal glucose-binding sites are similar to the ribokinase, while the specific glucose-binding sites for many bacterial glucokinases remain elusive. It turns out that the archaeal glucokinases, including the Aeropyrum pernix ATP dependent Glk, showed broad specificity for hexoses, such as fructose, mannose, glucosamine, N-acetylglucosamine, and N-mannosamine .
Glucokinases, which participate in carbon catabolite repression [10–13], contain the so-called ROK (repressor, ORF, kinase) [14, 15]. Two alternative ROK motifs have been suggested: [LIVM]X(2)G [LIVMFCT]GX [GA] [LIVMFA]X(8)GX(3–5) [GATP]X(2)G [R KH] [14, 15] and CXCGX(2)GX [WILV]EX [YFVIN]X [STAG] [9, 16]. Concha and Leon  further proposed cysteine (C) residues, especially those within the ROK motif, to be essential for the catalytic activity of glucokinase. These may also be required for glucose binding.
Here we describe the unexpected finding that the phylogenetic analysis provided two clusters of microbial glucokinases sequences, which are also distinguished by the presence or absence of the CXCGX(2)GCXE motif. Since B. subtilis GlcK contains this motif, the role of C residues within the motif as well as the remaining C residues was examined.
Glucose kinases/glucokinases (EC 126.96.36.199) comprise two lineages with or without a conserved CXCGX(2)GCXE motif
B. subtilis GlcK occurs in both monomeric and dimeric forms
The C175, C177, and C182 are essential for B. subtilis GlcK enzymatic activity
C321A mutation increases B. subtilis GlcK enzymatic activity, which maybe independent of the dimerization status
In order to confirm the enzymatic activity of GlcK mutants, we overproduced and purified the wild type GlcK, GlcKC166A, GlcKC282A, and GlcKC321A from soluble protein fractions of E. coli RB791. Induction of mutant GlcK was done with 1 mM IPTG at OD0.7. Three hours after induction, proteins were harvested and subjected to AKTA Purifier using Ni2+-NTA column. A 1000 ml culture yielded about 1 mg of pure GlcKC166A and about 10–15 mg of pure wild type GlcK, GlcKC282A, or GlcKC321A. The purified proteins were then tested for glucokinase activity in vitro, by coupling the phosphorylation of glucose to the formation of NADPH by glucose 6-phosphate dehydrogenese. The GlcKC166A activity was 12.3 ± 6.2 μmol min-1 (mg protein)-1, which was comparable to the wild type GlcK activity. The GlcKC282A activity, 26.0 ± 7.4 μmol min-1 (mg protein)-1, was slightly higher than GlcKC166A and the wild type GlcK. However, GlcKC321A's activity was 5-fold higher than GlcKC166A's and the wild type's. This result was in agreement with the in vivo functional complementation assay. Mutants GlcKC166A and GlcKC282A displayed red phenotype of E. coli UE26 colonies similar to the wild type GlcK (Fig. 4). As the enzymatic activity was much higher than that of the wild type, the C321A mutation caused much darker red colonies (Fig. 4). Similar to the wild type GlcK, SDS-PAGE analysis showed that GlcKC166A, GlcKC282A, and GlcKC321A appeared both as monomers and as homodimers under the non-reducing condition. The data suggests that the enzymatic activity of GlcK was independent of the dimerization status. Therefore, the increasing enzymatic activity of the GlcKC321A may not correlate well with the dimerization status. Nevertheless, whether the GlcK activity is affected by different ratios of monomer to homodimer warrants further study.
We have shown that two lineages of glucokinases has evolved with the presence or absence of the CXCGX(2)GCXE motif. Glucokinase belongs to the ROK family with the hallmark [LIVM]X(2)G [LIVMFCT]GX [GA] [LIVMFA]X(8)GX(3–5) [GATP]X(2) G [RKH] motif [14, 15]. Park et al., 2000  reported that some glucokinases do not contain the ROK motif. In fact, most glucokinase sequences, retrieved by us, preserved the ROK motif. However, some of the ROK motifs belonging to the second lineage have one to four amino acid mutations. Within the ROK family, the B. subtilis GlcK was grouped together with B. subtilis Xyl repressor protein (XylR), putative B. subtilis fructokinase (YdhR), Streptococcus mutans fructokinase (ScrK) encoded within a sucrose regulon, and Zymomonas mobilis fructokinase (FruK) [14, 15]. Dahl et al., 1995  analyzed the interaction of fructose, fructose 6-phosphate, glucose, and glucose 6-phosphate on the binding of XylR into xylO. Interestingly, only glucose stimulated the XylR binding . The XylR has a ROK motif and a CXCGX(2)GCXE motif. In contrast, Z. mobilis FruK, B. subtilis YdhR, and S. mutans ScrK contain ROK motifs but not the CXCGX(2)GCXE motif. Hence, the CXCGX(2)GCXE motif may correlate with glucose binding. This is a reasonable possibility considering that the GlcK mutants with cysteine substitution exhibited a loss of enzymatic activity (Fig. 4).
B. subtilis GlcK was present in both monomeric and dimeric forms (Fig. 3). Mutants GlcKC166A, GlcKC282A, and GlcKC321A were still able to form a homodimer as shown by SDS-PAGE, under non-reducing conditions. However, oxidation of GlcK led to the homodimer formation (Fig. 3B). The dimerization of GlcK is possibly due to the overall role of the ATPase domain. Proteins with the ATPase domain acquired the capacity to dimerize and bind to ATP in an active site between the two subunits [22–24]. The evidence for this comes from the overall structural symmetry between two domains of the ATPase as well as from the symmetric arrangement of the two phosphate binding loops . The ATPase domain of GlcK is located between amino acid residues 6 – 27: FAGIDLGGTTIKLAFINQYGEI (phosphate 1), 109 – 126: IENDANIAALGEMWKGAGDG (connect 1), 135 – 149: VTLGTGVGGGIIANG (phosphate 2), 255 – 282: PSKIVLGGGVSRAGELLRSKVEKTFRKC (adenosine), and 295 – 308: IAALGNDAGVIGGA (connect 2).
Multiple alignments and phylogenetic analysis had led directly to valuable insights into the possible molecular function and the evolution of glucokinase. This study enabled us to classify microbial glucokinases into two distinct lineages, with or without the CXCGX(2)GCXE motif. The experimental study also identified the role of C residues in B. subtilis GlcK. The three-conserved C residues in that motif are clearly essential for GlcK activity. However, the C321A mutation led to higher GlcKC321A enzymatic activity with respect to the wild type's, suggesting more adequate glucose phosphorylation.
Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study.
Strains and plasmids
Relevant genotype or phenotype
Reference or source
B. subtilis 168
F' [lacIq L8] hsdR+ hsdM
F- ptsG2 ptsM1 glk7 rpsL150
pQE9 derivative carrying glcK fused in frame to the His tag-coding region
pMD496 derivative carrying glcKD10K
pMD496 derivative carrying glcKC166A
pMD496 derivative carrying glcKC175A
pMD496 derivative carrying glcKC177A
pMD496 derivative carrying glcKC182A
pMD496 derivative carrying glcKC282A
pMD496 derivative carrying glcKC321A
Multiple sequence alignments and phylogenetic analysis
Sequences for eukaryotic, bacterial and archaeal glucokinases and putative glucokinases were retrieved from the GenBank database. Those sequences were aligned using the Clustal method  with the MegAlign 4.0 program (DNAStar Co., Madison, WI). To confirm the conservative domain, the obtained CXCGX(2)GCXE motif was used as template for BLAST searching . Neighbor joining distance trees of microbial glucokinases were produced using the phylogenetic package MEGA2 . Amino acid differences between sequences were corrected for multiple substitutions using a gamma correction. In this correction, α, the shape parameter of the gamma distribution, was set to 2. Therefore, the distance between any two amino sequences is approximately equal to Dayhoff's PAM distance per site . Support for the nodes within phylogenetic tree were evaluated by the bootstrap , which was done in 500 replicates of the whole data set.
Sets of primers used for site-directed mutagenesis in this study
5' tgcgggcatta ag ctgggaggaacgacgat 3'
5' aaccatatctcgtccatggatccgtgatgg 3'
5' ggccatattgc cagcatccctgaaggcgga 3'
5' gatttctccgccggcgccatttataccatg 3'
5' gcgcccgc caactgcggcaaaacgggctgt 3'
5' tccgccttcagggatgctgcaaatatggcc 3'
5' tgcaacgc cggcaaaacgggctgtatcgaa 3'
5' gggcgctccgccttcagggatgctgcaaat 3'
5' ggcaaaacgggcgc tatcgaaacaattgcg 3'
5' gcagttgcagggcgctccgccttcagggat 3'
5' cgcaaagc cgcgtttccgcgggcagcccaa 3'
5' gaatgttttctcgacttttgatctcagcag 3'
5' catcaaaatgc ttaaaattgtgtaaatgaa 3'
5' tttcagccattcatttttagcgatccaagc 3'
Pure plasmids, carrying either glcK or one of its mutants (Table 1), were prepared with the Nucleobond Midiprep kit according to the manufacturers suggestions (Macherey-Nagel, Dueren, Germany). Nucleotide sequences were determined by the cycle sequencing technique using the automated capillary sequencer, ABI PRISM 310/377 (Perkin Elmer Co., Foster City, CA). Sequencing primers used were: 5'CGGATAA CAATTTCACACAG3', 5'CTTCT GAGGTCATTACTGG3', 5'GCTGCGCTCGGGG AAATGTG3', and 5'GATACGCCGCCGCCAAGAAC3'. DNA analysis was carried out by DNAStar software (DNAStar Co., Madison, WI).
Protein overproduction and purification
Overexpression of [His]6-tagged-GlcK  and its mutated GlcKs was accomplished in E. coli RB791 harbouring the corresponding plasmids (Table 1). Cells were harvested three hours after induction with 0.1 mM IPTG at an OD600 of ~0.7. The pellet was then resuspended and sonicated in lysis buffer (150 mM NaCl and 20 mM Tris-Cl pH 7.5). Over-produced soluble proteins were purified from the supernatant as previously described . The crude extract of cells was quickly passed over a Ni2+-loaded HiTrap chelating column (Pharmacia, Freiburg, Germany), which had been equilibrated with 40 column volumes of washing buffer (200 mM NaCl, 20 mM Imidazole and 5 mM Tris-Cl pH 7.5). Pure protein was eluted by a linear gradient using elution buffer (200 mM NaCl, 500 mM Imidazole and 5 mM Tris-Cl pH 7.5) at a flow rate of 0.5 ml min-1. Eluted protein aliquots of 0.5 ml were analysed on 12% SDS-PAGE. The GlcK concentration was determined by absorption measurement at 280 nm in 50 mM Tris-Cl pH 7.
In vivo functional complementation of B. subtilis GlcK mutants in E. coli UE26
E. coli strain UE26 (ptsG ptsM glk) was transformed with plasmids carrying wild type glcK or glcK mutants. In vivo glucokinase activities were observed by monitoring colonies' colour shift from white to red on MacConkey agar. The agar was supplemented with 50 mM glucose and 100 mM fucose .
In vitro assay of glucokinase activity
Enzymatic activity of wild type or mutated GlcK was quantified in vitro by a method described previously . Specific glucokinase activity was determined in a coupled enzyme assay by the method of Seno and Charter  in a solution consisting of 50 mM Tris-HCl pH 7.5, 20 mM glucose, 25 mM MgCl2, 0.5 mM NADP, 1 mM ATP, and 1 U of glucose 6-phosphate dehydrogenase (G6PDH). The G6PDH activity was assayed by monitoring the change in the optical density at 340 nm at 32°C with NADP as a cofactor.
Analysis of protein multimerization with SDS PAGE, oxidative cross-linking, and MALDI-TOF mass spectrometry
GlcK was subjected to reducing or non-reducing conditions by using loading buffer (0.1% Bromphenolblue, 16% Glycerol, 4% SDS, and 55 mM Tris-Cl pH 6.8) with or without 10% β-mercaptoethanol. The samples were analysed on 12% SDS-PAGE. Oxidative cross-linking was carried out either with H2O2 or with a complex of Cu(II) and 1,10-phenantroline. The procedure for the oxidative cross-linking of glucokinase was carried out as previously described  using 10 μg of glucokinase and analysing using an 8% SDS-PAGE. In order to remove the reductant, samples were dialyzed for several hours at 4°C in buffer (5 mM Tris-Cl pH 8.4, 1 mM EDTA and 1 mM DTT) containing 8 M, 5 M, or without urea . Multimerization and molecular mass determination of B. subtilis GlcK was performed on a Biflex™ III Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany) equipped with a nitrogen laser at λ = 337 nm at the Institute for Biochemistry, University of Erlangen-Nuremberg.
Fluorescence studies, carried out on a Spex Fluorolog spectrometer (Edison, NJ, USA), were used to determine spectral changes of GlcK and its mutants. The excitation wavelength was set to 280 nm or 295 nm and the emission was recorded in the range of 300 nm to 450 nm. For these measurements, the slit widths were set to 2.2 mm. Fluorescence measurements were carried out at 22°C.
This paper is dedicated to Dr. Michael K. Dahl, who passed away on May 4, 2003. We are grateful to U. Ehmann, S. Schoenert, P. Schubert, and T. Buder for their interest and help. We would like to thank T. Bonk at the Institute for Biochemistry, University of Erlangen-Nuremberg for assistance with the MALDI-TOF MS and B. Scott of the Faculty of Medicine, University of Ottawa, for critical reading of the manuscript. The experimental work was carried out in the laboratories of W. Hillen (University of Erlangen-Nuremberg) and W. Boos (University of Konstanz).
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