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Targeting Acetate Kinase: Inhibitors as Potential Bacteriostatics
Targeting Acetate Kinase: Inhibitors as Potential Bacteriostatics
Journal of Microbiology and Biotechnology. 2013. Nov, 23(11): 1544-1553
Copyright © 2013, The Korean Society For Microbiology And Biotechnology
  • Received : May 13, 2013
  • Accepted : August 06, 2013
  • Published : November 28, 2013
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About the Authors
Saeme Asgari
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran
Parvin Shariati
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
aehabibi@sina.tums.ac.ir
Azadeh Ebrahim-Habibi
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran
aehabibi@sina.tums.ac.ir

Abstract
Despite the importance of acetate kinase in the metabolism of bacteria, limited structural studies have been carried out on this enzyme. In this study, a three dimensional structure of the Escherichia coli acetate kinase was constructed by use of molecular modeling methods. In the next stage, by considering the structure of the catalytic intermediate, trifluoroethanol (TFE) and trifluoroethyl butyrate were proposed as potential inhibitors of the enzyme. The putative binding mode of these compounds was studied with the use of a docking program, which revealed that they can fit well into the enzyme. To study the role of these potential enzyme inhibitors in the metabolic pathway of E. coli , their effects on the growth of this bacterium were studied. The results showed that growth was considerably reduced in the presence of these inhibitors. Changes in the profile of the metabolic products were studied by proton nuclear magnetic resonance spectroscopy. Remarkable changes were observed in the quantity of acetate, but other products were less altered. In this study, inhibition of growth by the two inhibitors as reflected by a change in the metabolism of E. coli suggests the potential use of these compounds (particularly TFE) as bacteriostatic agents.
Keywords
Introduction
Acetate kinase, a conserved enzyme that is widespread in bacteria and archaea, is responsible for the phosphorylation of acetate [15 , 16] . Since acetyl coenzyme A is a precursor of fatty acid synthesis, it is acceptable that primary cellular growth depends on the activation of acetate through the process of intracellular phosphorylation.
Acetate kinase has different roles in bacteria and archaea [2 , 5 , 6 , 17] . The primary bacteria use acetate kinase for energy generation by converting ADP and acetyl phosphate to ATP and acetate. Archaea use the reverse of this reaction as the first step of methanogenesis, the sequestration and activation of acetate [3 , 9 , 19 , 23] .
Further studies on acetate kinase structure will undoubtedly reveal more information regarding its mechanism of action, and since it is important in both catabolism and anabolism in many bacteria and some eukaryotes, inhibitors of this enzyme may have potential antibacterial effects.
A common approach to designing kinase inhibitors has been to target the ATP binding sites of these enzymes because such inhibitors possess certain structural similarities with ATP [28] . However, potentially selective inhibitors could be designed based on the structure of the substrate and binding sites of kinases.
The study and analysis of protein sequences play an important role in identifying biological processes and may be useful in designing new proteins and drugs and predicting the structure and function of proteins [18 , 20] . As of 2011, the three-dimensional structures of acetate kinase have been resolved for Thermotoga maritima, Methanosarcina thermophila , and Mycobacterium avium .
Based on the X-ray crystallographic studies, it has been shown that seven protected amino acids comprising Asn 7 , Arg 91 , His 123 , Asp 148 , His 180 , Arg 241 , and Glu 384 are important for acetate kinase activity in M. thermophila [1 , 7 - 9] . These amino acids are also conserved in the acetate kinases of eukaryotes [9 , 10 , 19 , 24] .
In the present study, two ligands, namely 2,2,2- trifluoroethanol (TFE) and 2,2,2-trifluoroethyl butyrate (TFEB), were proposed as potential inhibitors of acetate kinase based on the structure of the acetate molecule. A molecular model of the E. coli enzyme was generated, and after in silico testing of ligands in the model, they were then checked experimentally with respect to the growth rate of E. coli . The results obtained in this study could be of potential use in the design of more potent bacteriostatics.
Materials and Methods
- Homology Modeling
The ClustalW program was used for multiple sequence alignments of the acetate kinase from E. coli , M. thermophila , T. maritima , and M. avium [13] . A homology model was built for the acetate kinase of E. coli using the ModWeb server (ModWeb version SVN.r1368M) [4 , 22] , with T. maritima (PDB code 2IIR) used as a template. ModWeb uses the MODELLER program to compute homology models. Models are generated based on the satisfaction of spatial restraints of the target sequence, which is aligned with the 3D structure of the template. The spatial restraints are expressed as probability density functions and are related to a function optimized by use of conjugate gradients and molecular dynamics with simulated annealing; the stereochemical restraints ( e.g ., bond length and bond angle) preferences are based on the CHARMM molecular mechanics forcefield. The program also incorporates limited functionality for ab initio structure prediction of loop regions of proteins, which are often highly variable even among homologous proteins [4] .
The sequence similarity between the template and the model sequences was 70%. The quality of the model was verified using the Procheck_NT program [14] . The MOE 2010.10 program (Chemical Computing Group Inc., Montreal, Canada) was used for the visualization and comparison of structures. Topologies of model and template structures were generated by use of the Pro-origami system [25] .
- Ligand Search and Docking
The Web interface of the ligand similarity search, F-trees ( http://public.zbh.uni-hamburg.de/ftrees/query.py ), was used to find molecules similar to acetyl phosphate. The selected compounds, 2,2,2-trifluroethanol and 2,2,2-trifluroethyl butyrate, were then docked into the active site of the E. coli acetate kinase model using the Autodock Vina program [26] . Exhaustiveness was set to 20, and a computer with eight processors was utilized for the computation; 100 poses were generated for each ligand. Visualization of the most probable interactions was done by use of LIGPLOT + [31] .
- Microorganisms and Cultivation Conditions
The bacterial strains used in the present study were E. coli PTCC 1399, Pseudomonas aeruginosa PTCC 1431, and Staphylococcus aureus PTCC 1430. The strains were precultured in shake flasks containing Luria-Bertani (LB) medium consisting of 10 g of peptone, 5 g of yeast, 5 g of NaCl, and 40 g of glucose per liter. The cultures were incubated at 37℃ with shaking at 150 rpm. A 1 ml sample of the growth cultures was then transferred to 100 ml of M9 medium containing 0.4% glucose as the sole carbon source, and cultivation was continued at 37℃ in shake flasks at 150 rpm. For anaerobic growth, the same conditions as above were used; however, a CO 2 incubator was utilized instead of the shaker. Cell density was measured spectrophotometrically at 600 nm [11 , 21 , 27] .
- NMR Analysis of Metabolic End-Products
Escherichia coli cells were grown in M9 medium (pH7.2) containing 0.4% glucose. When the cell optical density reached approximately 1 at 600 nm (in the logarithmic phase), the cells were then collected by centrifugation at 5,900 × g for 3 min at 4℃. The supernatant was discarded and the cell pellets were suspended in M9 medium without glucose and washed twice with the same solution. The cells were finally suspended in M9 medium (pH7.2) containing 0.4% glucose until a cell optical density of approximately 1 was achieved at 600 nm. In order to study the products of this metabolism, the cell suspension was incubated separately with TFE (0.13 M) and TFEB (0.13M) at 37℃ for 4-5 h (ligand volume and concentration were adjusted based on molecular weight and density). A 0.9 ml sample of the cell suspension containing 0.1 ml of D 2 O was placed in a 5-mm-diameter NMR tube. Proton NMR spectra were then obtained at 500 MHz [12] .
Results
- Molecular Modeling Studies
Comparison of the E. coli acetate kinase structure with reported three-dimensional structures. An alignment of the E. coli enzyme sequence with sequences of enzymes that possess an established three-dimensional structure in the protein databank (PDB) is shown in Fig. 1 . In the three sequences for which three-dimensional data exist, it was observed that the seven amino acids, His 123 , His 180 , Phe 179 , Val 93 , Arg 91 , Pro 232 , and Arg 241 , which have been shown to be important in acetate binding of the M. thermophila enzyme [9 , 10 , 19] , are also conserved. These residues are His 122 , His 179 , Phe 178 , Val 92 , Arg 90 , Pro 231 , and Arg 240 in T. maritima and His 113 , His 170 , Phe 169 , Val 83 , Arg 81 , Pro 222 , and Arg 231 in M. avium . In the E. coli protein sequence, these amino acids are also conserved, except for Phe 179 , which is replaced with Ala 181 (His 123 , His 182 , Ala 181 , Va l93 , Arg 91 , Pro 234 , Arg 243 ). These amino acids are mostly conserved in all of the reported sequences in the NCBI protein database; the differences are mainly related to the two amino acids proline and valine. In certain species ( e.g., Entamoeba ), Met is found instead of Pro, and in some other species ( e.g., Archeobacter ), Val is replaced with Ala in the active site.
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Alignment of the E. coli acetate kinase enzyme sequence with those of T. maritima, M. thermophila, and M. avium. Conserved regions are marked with an asterisk. Color codes of amino acids are as follows: residues A, V, F, P, M, I, L, and W (small + hydrophobic (incl. aromatic -Y)) in red; residues D and E (acidic) in blue; residues R and K (basic) in magenta; residues S, T, Y, H, C, N, G, and Q (hydroxyl + sulfhydryl + amine + G) in green.
Amino acids that are present in the ADP binding sites are also considerably conserved in different species, but less remarkably than the acetate binding sites. In some cases, an amino acid with similar properties has replaced the original one. For example, in the Escherichia, Lactobacillus, Haemophilus, Streptococcus , and Yersinia species, Phe 179 has been replaced by alanine, and in Vibrio cholera , Phe179 is substituted by Met177. Of course, substitution of Pro 232 by Thr in Entamoeba histolytica could be considered a significant change, as is Phe 284 , which is substituted by Cys in some sequences. In another example, Ile 332 is seen in a number of sequences, while in some ( Flavobacter, Streptococcus ), it has been substituted by Met, which occupies a greater volume, thus resulting in less available space within the active site. On the other hand, amino acids that are involved in direct interaction with acetate are more protected ( e.g ., His 123 and Arg 241 ). Differences are greater for the binding site of ADP in which more amino acids are involved.
Homology modeling. To obtain data regarding the relationship between the structure and function of acetate kinase, a model of the three-dimensional structure of the E. coli acetate kinase was generated by homology modeling. Among X-ray crystallographic structures that have been reported for acetate kinase, the T. maritima enzyme structure was used as the template, based on the fact that it has the highest sequence similarity with the target enzyme (70%). The Modeller Web server was used to generate the model, the overall secondary structure of which is shown in Figs. 2 A and 2 B, relative to the template structure ( T. maritima enzyme, Figs. 2 C and 2 D). Quality evaluation of the model made with Procheck ( Table 1 ) shows that 95.4% of the amino acids are positioned at the core (best) region, and only one residue is found in the disallowed region (Asp 322 ). When comparing the template and model enzymes, it was found that the T. maritima enzyme also contains an aspartate residue at this position, but in the model enzyme, the surrounding residues are different, which may affect the dihedral angles of Asp 322 . However, this residue is located far from the active site and is not part of the conserved residues.
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Secondary structure components of the E. coli acetate kinase model (A, B) as well as the T. maritime acetate kinase (C, D).
Statistics of the model protein Ramachandran plot, as obtained by use of the Procheck program.
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Statistics of the model protein Ramachandran plot, as obtained by use of the Procheck program.
A comparison of the topologies of the E. coli model and T. maritima indicates an overall similar structure, with the principal differences being the lengths of sheets 1, 2, 5, and 32 as well as helices 21, 23, 24, and 27. The sheets and helix 24 are shorter in the model, whereas helices 21, 23, and 27 are larger, as compared with those of the template. When acetate is docked into the monomeric acetate kinase model by the Autodock program, a binding mode can be obtained that conforms to the acetate binding site in the template enzyme ( Fig. 3 A) . According to this result, the main interaction between the acetate molecule and enzyme would occur via the hydrogen bonds of Gly 180 and Gly 231 . Accordingly, the backbones of these residues are interacting with the oxygen atom in the acetate molecule. A number of hydrophobic interactions are also observed that involve Tyr 179 , Ala 181 , Met 230 , Leu 232 , Pro 234 , and Thr 233 . It should be noted that in the template that has been used, no ligand has been cocrystallized in the active site; however, another bacterial enzyme structure, namely acetate kinase from M. thermophila , has been reported to contain acetate, ADP, and the transition state analog AlF 3 . The binding mode of these ligands is indicated in Fig. 3 B. In fact, the binding site of acetate is similar to what we have obtained ( e.g ., Gly 178 , which is equivalent to Gly 180 in this study, as seen in the vicinity of acetate), and the slight difference in the positioning of acetate is attributable to the presence of the additional AlF 3 molecule.
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Schematic representation of the interactions of docked acetate in the E. coli acetate kinase enzyme. (A) Location of acetate, AlF3, and ADP in M. thermophila acetate kinase from the 1TUY.pdb file (B), and interactions of the docked ligands TFE (C) and TFEB (D) with the E. coli acetate kinase enzyme, as obtained by the docking experiment. Images were obtained by the use of Ligplot+ (A, C, D), and MOE.2010.10 (B). In the Ligplot+ schemes, hydrogen bonds are indicated by dashed lines between the atoms involved, and hydrophobic interactions are represented by an arc with spokes radiating toward the ligand atoms they contact. These atoms are shown with spokes radiating back [31].
Proposal for inhibitors: similarity search and docking. Following the primary studies of the acetate kinase structure, the next step was to find potential inhibitors of the acetate kinase enzyme, based on the acetate binding site. The F-trees tool was used as a means to provide some preliminary ideas about putative ligands based on their similarities to the acetyl phosphate structure. From the results that were obtained, TFE and TFEB were chosen as simple structures that could also be tested experimentally. Autodock Vina was then used to study the potential mode of insertion of these structures in the acetate kinase enzyme. The best binding energies obtained for the ligands were as follows: acetate, -3 kcal/mol; TFE, -3.7 kcal/mol; and TFEB, -4.1 kcal/mol. The TFE and TFEB positions corresponding to these results are shown in Figs. 3 C and 3 D, respectively. Both of these molecules occupy a wider volume compared with acetate, and could make more putative interactions with nearby amino acids located in the active site. Interestingly, the binding mode obtained for TFE is highly similar to that of acetate (as explained above), whereas the TFEB interactions are only hydrophobic ones, which take place with the Val 93 , His 94 , His 123 , Thr 151 , Gly 180 , Ala 181 , and Pro 234 residues.
- Experimental Studies
Effects of TFE and TFEB on the growth of E. coli. To validate the results obtained by the modeling tests, the effects of TFE and TFEB on bacterial growth were studied. According to preliminary experimental studies, bacterial growth was reduced in the presence of these inhibitors ( Figs. 4 A- 4 D). Similar results were obtained under both aerobic and anaerobic conditions ( Figs. 4 A- 4 D). The growth of two other bacterial strains, one gram-negative ( Pseudomonas aeruginosa ), and one gram-positive ( Staphylococcus aureus ), was also tested in the presence of TFE to determine the potential range of its effects. As shown in Figs. 4 E and 4 F, TFE was able to reduce the growth of both species under aerobic conditions but had a more pronounced effect on P . aeruginosa .
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Comparison of growth rates by measuring absorbance at 600 nm. (A) Anaerobic control sample without inhibitor (●) and in the presence of TFE (■), (B) aerobic control sample without inhibitor (●) and in the presence of TFE (■), (C) anaerobic control sample without inhibitor (●) and in the presence of TFEB (■), (D) aerobic control sample without inhibitor (●) and in the presence of TFEB (■), (E) aerobic control sample of Pseudomonas aeruginosa without inhibitor (●) and in the presence of TFE (■), and (F) aerobic control sample of Staphylococcus aureus without inhibitor (●) and in the presence of TFE (■).
Study of the metabolites produced by bacterial cells in the presence and absence of inhibitors. In order to further analyze the preliminary experimental results and study the mechanism of the effect of the inhibitor on bacterial growth, more precise and sensitive determinations were made by NMR analysis of metabolites produced in the presence and absence of the two inhibitors. NMR spectra of the metabolites produced by the growth of E. coli in the presence and absence of TFE and TFEB indicate that the inhibitors clearly affect the production of the metabolites acetate, ethanol, lactate, pyruvate, and formate by this bacterium ( Figs. 5 A- 5 C).
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NMR scans of glucose fermentation under anaerobic conditions. (A) Culture was incubated in minimal medium with glucose (control sample). (B) Culture was incubated in minimal medium with glucose in the presence of TFE. (C) Culture was incubated anaerobically in minimal medium with glucose in the presence of TFEB. The metabolic products are ethanol (E), lactate (L), acetate(A), pyruvate (P), and formate (F).
The main product produced under aerobic conditions was acetate, and negligible amounts of ethanol, lactate, pyruvate, and formate were also observed ( Figs. 6 A- 6 C).
Rates of growth were reduced in the presence of the inhibitors under both aerobic ( Figs. 4 B and 4 D) and anaerobic ( Figs. 4 A and 4 C) conditions. Such a reduction was due to a decrease in the production of acetate and other metabolites by E. coli in the presence of the TFE inhibitor. Ethanol and formate levels were also reduced, but lactate levels increased even though pyruvate concentrations had not changed significantly ( Figs. 5 B and 6 B).
Similar observations were also made in the presence of the TFEB inhibitor, whereby acetate and ethanol were also reduced but formate levels did not change significantly ( Figs. 5 C and 6 C). Lactate levels increased more under anaerobic conditions owing to anaerobic growth and acidic conditions ( Figs. 6 B and 6 C).
Based on the results obtained from the NMR studies, it is possible to suggest that these compounds influence the production of bacterial metabolites directly or indirectly by affecting acetate kinase and/or enzymes related to acetate kinase.
Both TFE and TFEB clearly cause a reduction in the levels of acetate, growth yield, and rates of growth in E. coli . In fact, TFE was more effective than TFEB, and hence can be considered as a bacteriostatic against E. coli and other facultative anaerobic bacteria.
Inhibitors that are designed for kinase enzymes generally target the ATP binding sites [28] , whereas the substrate binding sites of kinases have not been previously shown to be the target of inhibitors. The present study shows that it is possible to design inhibitors for the substrate (acetate) binding site. Considering the role of this enzyme in bacterial metabolism, its inhibition may have at least a bacteriostatic effect.
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NMR scans of glucose fermentation under aerobic conditions. (A) Culture was incubated in minimal medium with glucose (control sample). (B) Culture was incubated in minimal medium with glucose in the presence of TFE. (C) Culture was incubated in minimal medium with glucose in the presence of TFEB. The metabolite products are ethanol (E), lactate (L), acetate (A), pyruvate (P), and formate (F).
Discussion
One of the major products of anaerobic fermentation of glucose by E. coli is acetate. Acetyl-CoA of the glycolytic pathway is converted to acetyl phosphate by phosphotransacetylase in a phosphorylation reaction. Acetyl phosphate is subsequently converted to acetate by acetate kinase. During this reaction, the high energy phosphate of acetyl phosphate is transferred to ADP, generating a single ATP molecule. In E. coli , the conversion of acetyl phosphate to acetate provides a major source of ATP during anaerobic growth [12] .
In this study, proton NMR spectroscopy was carried out to observe the effects of two compounds, TFE and TFEB, on the growth and fermentation of glucose by E. coli . NMR analysis of culture supernatants was carried out following incubation of E. coli cells with glucose in the presence and absence of the two inhibitors under both aerobic and anaerobic conditions. Under aerobic conditions, the major product formed was acetate, followed by smaller concentrations of ethanol, lactate, pyruvate, and formate. The formation of large amounts of acetate was consistent with the anticipated transfer of electrons to oxygen via the aerobic respiratory chain. However, when cells were incubated with either of the two inhibitors in the presence of glucose, lower rates of growth were observed when compared with growth with glucose only. In fact, lower rates of growth were achieved in the presence of TFE when compared with those in the presence of TFEB. Such reductions in growth were reflected by a decrease in acetate levels and the other metabolic products. In the presence of TFE, the concentrations of ethanol, acetate, and formate were reduced 2-fold. However, pyruvate levels did not change. A similar situation was also seen in the presence of TFEB; however, reductions in acetate levels were not as drastic as in the case of TFE, and formate levels did not change significantly. Interestingly, the effect of TFE seems to be a generic one under aerobic conditions since the compound was able to inhibit the growth of two other bacterial strains.
A similar trend was also observed under anaerobic conditions. However, in the presence of glucose under anaerobic conditions, the usual mixed-acid fermentation products were formed (ethanol, lactate, acetate, pyruvate, and formate) [12] . Larger amounts of ethanol and pyruvate were produced when compared with aerobic conditions. Higher levels of lactate induced by acidic and anaerobic conditions were also produced. The NMR spectra of anaerobic glucose fermentation demonstrated the typical ratio of ethanol/acetate remaining as approximately 1:1(or50:50)In this way, the redox and fermentation balance are maintained. Once again, the inhibitor TFE was more effective than TFEB, resulting in an approximately 2-fold decrease in acetate levels and larger reductions in growth yield and rate. However, in contrast to aerobic conditions, pyruvate levels doubled in the presence of TFEB. It seems that under anaerobic conditions, TFE is again more effective than TFEB in reducing acetate levels and rates of growth. It may be that TFE acts at the enzyme or genetic level, inhibiting or lowering the activity of certain enzymes at key points in the fermentation pathway. One such enzyme that it may act upon is pyruvate formate lyase (PFL), which is responsible for the production of acetyl-CoA and formate from pyruvate. It should be noted that the acetyl- CoA produced by PFL is also subsequently converted to ethanol and acetate. Hence, it is highly possible that TFE is effective at this stage of the glucose fermentation pathway, with a possible reduction in activity or inhibition of the key fermentation enzyme PFL. This inevitably leads to the generation of less acetate and ATP, thus causing reduction in growth yield and rate. The same may be explained for TFEB, but with a weaker response.
Another plausible explanation may be more direct, wherein TFE acts directly upon acetate kinase or phosphotransacetylase, thus leading to a reduction in acetate formation. In fact, in a previous study, an E. coli strain lacking the key enzymes acetate kinase and phosphotransacetylase of the major acetate synthesis pathways, reduced acetate accumulation and also exhibited an increased lactate synthesis rate [29 , 30 , 32] .
These observations should be further investigated in the future by carrying out appropriate enzyme assays and studying the effect of such inhibitors at the molecular genetic level.
Another possible explanation for such reductions in acetate and growth levels may simply be due to competition between acetyl-CoA and the products from the metabolism of the inhibitor for reducing equivalents generated by glucose metabolism. However, this may be unlikely because it is still uncertain if alcohol inhibitors such as TFE can be broken down by E. coli .
It should be noted that a factor that greatly affects the fermentation product ratio is the type and redox level of the substrate. Thus, sugar alcohols, which are more reduced than glucose, yield a higher proportion of the more reduced fermentation products such as ethanol. Those more oxidized than glucose (hexonic and hexuronic acids) yield more of the oxidized products, such as acetate. In this way, the bacterium can maintain redox balance [12] .
In conclusion, both compounds TFE and TFEB act to reduce acetate production and growth yields and rates in E. coli , with TFE being more effective than TFEB. These results demonstrate that TFE can be considered as a potential bacteriostatic agent with an ability to act against pathogenic facultative anaerobes, such as E. coli , infecting the human body.
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