[The preparation and identification of diagnostic recombinant glycerol kinase].

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase.

In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His(6) tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 A resolution indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 A. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V(M)) of 2.5 A(3) Da(-1), corresponding to 50% solvent content.

Structural characterizations of glycerol kinase: unraveling phosphorylation-induced long-range activation.

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ("thermal factors") in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.

Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region.

The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degrees C which was 3.1 degrees C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.

Subcloning, expression, purification, and characterization of Haemophilus influenzae glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia coli is an allosteric regulatory enzyme whose activity is inhibited by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously termed III(Glc)). Comparison of its primary structure with that of the highly similar Haemophilus influenzae glycerol kinase reveals that the amino acid sequence for the binding site for FBP is conserved while the amino acid sequence for the binding site for IIA(Glc) contains differences that are predicted to prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was assembled from DNA library fragments and subcloned into pUC18. The enzyme is expressed at high levels in E. coli. It was purified to greater than 90% homogeneity by taking advantage of its solubility behavior in a procedure that requires no column chromatography. The initial-velocity kinetic parameters of the purified enzyme are similar to those of the E. coli glycerol kinase. The H. influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement with the prediction based on sequence comparison. Sedimentation velocity experiments reveal that inhibition of HiGK by FBP is associated with oligomerization, behavior which is similar to EcGK. The possibility of utilizing mutagenesis studies to exploit the high degree of similarity of these two enzymes to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed.

Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei.

We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus.

The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.

Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.

A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure.

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.

Purification and characterization of glycerol kinase from Trypanosoma brucei.

Glycerol kinase (EC 2.7.1.30)(GK) from the glycosomes of Trypanosoma brucei has been purified and its kinetic properties have been examined. It has a molecular weight of approximately 53,000 and exists in solution as a monomer. This GK has a broad pH optimum, with equal activity between pH 7 and 9.5. Its catalytic mechanism appears to be random bi bi, with some cooperativity in substrate binding at high pH. The apparent Michaelis constants are: Kglycerol = 0.26 +/- 0.02 mM and KATP = 0.19 +/- 0.02 mM at pH 7.4, and Kglycerol = 0.17 +/- 0.03 mM and KATP = 0.26 +/- 0.02 mM at pH 9.0. Glycerol-3-phosphate (G3P) up to 10 mM displays virtually no product inhibition of the forward reaction, but ADP is a weak inhibitor, competitive with ATP and uncompetitive with glycerol. The forward reaction is catalyzed very efficiently in vitro, but the reverse reaction proceeds at an extremely low rate, consistent with its unfavorable delta G. Under anaerobic conditions T. brucei GK is thought to convert ADP and G3P to ATP and glycerol rapidly inside the intact glycosome, where it is tightly coupled to the other glycosomal enzymes. Our kinetic analyses suggest that GK may not rely on any unusual intrinsic properties to catalyze this reverse reaction: rather, the unusually high intraglycosomal concentrations of G3P and ADP, and the presence of efficient ATP traps, may drive this reaction by mass action.

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties.

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).

Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei.

A method is presented for the simultaneous purification of hexokinase, fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase, and the partial purification of glycerol-3-phosphate dehydrogenase (NAD+), 6-phosphofructokinase, glucosephosphate isomerase, and glycerol kinase from Trypanosoma brucei. As a first step, the glycosomes, microbody-like organelles of Trypanosomatidae, containing almost exclusively enzymes involved in glucose and glycerol metabolism [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364], were purified eightfold from homogenates with an average yield of 38%. Subsequently, the glycosomal content was subjected to hydrophobic interaction chromatography on phenyl-Sepharose. This step results in pure hexokinase (15% final yield) and almost pure triosephosphate isomerase, while the other glycosomal enzymes elute as mixtures of two or three enzymes. Triosephosphate isomerase was further purified to homogeneity on CM-cellulose (33% final yield), while phosphoglycerate kinase and fructose-bisphosphate aldolase were separated from each other and purified to homogeneity by affinity chromatography using ATP-Sepharose (25% and 30% final yields, respectively). Fructose-bisphosphate aldolase was further characterized as a typical class I enzyme.

Human and rat adrenal glycerol kinase: subcellular distribution and bisubstrate kinetics.

The subcellular distribution of adrenal glycerol kinase in man and rat are reported and the bisubstrate kinetics of the soluble enzyme are compared in these two species. The specific activity of glycerol kinase in human whole adrenal homogenate (145 microU/mg protein) was 3 times that found in rat whole adrenal homogenate (48 microU/mg protein). In both species 8% of the total glycerol kinase activity was associated with the nuclear pellet fraction. In human, 62% of the total activity was soluble, while 24% was associated with the postnuclear particulate fraction. Rat glycerol kinase activity was also predominantly soluble: 69% of the total activity was soluble and 13% was in the postnuclear particulate fraction. The apparent Km for glycerol in soluble adrenal glycerol kinase was similar in both species, 2.8 microM in human and 3.1 microM in rat. The apparent Km for ATP in soluble human adrenal glycerol kinase was 22.0 microM. In rat the enzyme did not appear to follow Michaelis-Menten kinetics with ATP as substrate. The Vmax for the soluble enzyme was similar in both human and rat. This report provides a background to biochemical investigations on human glycerol kinase deficiency, an inborn error of metabolism which may be characterized by adrenal hypoplasia and insufficiency.

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus.

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.

Studies on glycerol kinase and its role in ATP synthesis in Trypanosoma brucei.

Glycerol kinase of Trypanosoma brucei has been shown to be capable of catalysing sn-glycerol-3-phosphate dependent ADP phosphorylation for ATP generation. The rate of this reaction (Vr) is sufficient to account for the observed rate of glycerol production from anaerobic glucose metabolism by intact cells and to account for net ATP synthesis. Glycerol kinase has been purified by preparing a post-nuclear, particulate fraction and solubilizing the enzyme with 0.5% (w/v) Triton X-100. This treatment results in a 3.5-fold increase in total activity, demonstrating the latent nature of particulate glycerol kinase, and an overall 10-fold increase in specific activity in the soluble fraction. The ratio of the velocities of the forward (Vf) reverse (Vr) reactions of this enzyme is altered from 21 to 170 upon solubilization. The Michaelis constants for the solubilized enzyme are KmADP = 0.12 +/- 0.04 mM, KmG-3-P = 5.12 +/- 1.47 mM, Kmglycerol = 0.12 +/- 0.05 and KmATP = 0.19 +/- 0.04 mM. Endogenous hexokinase acts as an ATP trap favouring ATP synthesis sn-glycerol-3-phosphate and ADP. This can be demonstrated in reconstituted systems using trypanosome glycerol kinase and varying hexokinase activities. Mass action inhibition of ATP synthesis by glycerol is more marked with lower hexokinase activities. High glycerol kinase activity (> 0.5 mumol/min/mg protein) has been found in the T. brucei complex of trypanosomes that produce glycerol anaerobically whereas only low activities (less than or equal to 0.03 mumol/min/mg protein) are present in Trypanosoma cruzi, Trypanosoma lewisi and Crithidia fasciculata, organisms that do not produce glycerol. Trypanosoma congolense has a glycerol kinase activity of 0.17 mumol/min/mg protein and shows poorer ATP synthesis from anaerobic glucose metabolism than organisms of the T. brucei complex.

Chromatography in presence of high concentrations of salts on columns of celluloses with and without ion exchange groups (hydrogen bond chromatography). Its application to purification of yeast enzymes.

1. All the water-soluble yeast enzymes tested, which were only partially precipitated at best in the presence of high concentration of salts such as ammonium sulfate or sodium formate, were adsorbed on a column of cellulose in the presence of the same concentrations of the salts, and the adsorbed enzymes were chromatographically eluted by decreasing the concentration of the salts. 2. Even in the presence of high concentration of the salts, the adsorbed enzymes were eluted by urea or by "hydroxy-rich" reagents such as sucrose. 3. Under the experimental conditions used, the salt concentrations required for elution of the adsorbed enzymes were lower with cellulose than with DEAE-cellulose, CM-cellulose, or P-cellulose, indicating that ion exchange groups, either cationic or anionic, affected the adsorption, although the ion exchange groups of DEAE-cellulose, CM-cellulose, and P-cellulose were weakly but definitely functional as ion exchangers even in the presence of high concentrations of the salts. 4. The principal attractive force between cellulose and the enzyme was deduced to be due to hydrogen bonding. 5. This hydrogen bond chromatography was applied for the purification of some yeast enzymes.