Enolpyruvyl Transferase Research Articles

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium.

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays.

UDP-N-Acetylglucosamine enolpyruvyl transferase (MurA) of Acinetobacter baumannii (AbMurA): Structural and functional properties

Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target.

Computed insight into a peptide inhibitor preventing the induced fit mechanism of MurA enzyme from Pseudomonas aeruginosa.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the key enzymes involved in peptidoglycan biosynthesis. The peptide HESFWYLPHQSY (called PEP 1354) is an inhibitor of MurA with an IC50 value of 200μm. In this article, we have used the FlexPepDock ab-initio protocol from the Rosetta program homology modeling and molecular dynamics simulations to analyze, for the first time, the interaction of the PEP 1354 peptide with MurA enzyme from Pseudomonas aeruginosa (MurA-PA). Our modeling results suggest that the peptide binds to the same active site as the natural substrate UDP-N-acetylglucosamine (UNAG). Additionally, the MurA-peptide complex revealed that the peptide seems to prevent the closure of the Pro114-123 loop and, consequently, the open-closed transition of the MurA structure.

Coprinuslactone protects the edible mushroom Coprinus comatus against biofilm infections by blocking both quorum-sensing and MurA.

Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared with planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria ontheir sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3,4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin andrhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications.

English

Enterobacter cloacae is a clinically significant Gram-negative, facultatively-anaerobic, rod-shaped bacterium belonging to the family of Enterobacteriaceae. It has emerged as a prevalent nosocomial pathogen due to high level resistance to disinfec- tants and antimicrobial agents. The availability of complete genome sequence of E. cloacae has paved the new way to identify the novel drug targets. In the present work comparative analysis of the metabolic pathways of the pathogen and host was performed to identify the novel drug targets involved in pathogen but absent in Homo sapiens. All enzymes involved in the metabolic pathways of E. cloacae were searched against the proteome of H. sapiens using the BLASTp program. The threshold of percentage identity was set to as 0.001, and the query coverage < 50. Using these parameters approximately 44 unique putative targets were identified. Out of those non-homologous targets, 22 coding genes for putative targets were identified as essential genes from the DEG database. Based on extensive literature search, 8 targets such as UDP-N-acetylmuramoyl-L-alanyl-D-gluta- mate synthetase, UDP-N-acetylmuramate-L-alanine ligase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6-diaminopimelate ligase, UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase, UDP-N-acetylmuramate-L-alanine ligase (MurC) α-isopro- pylmalate synthase, UDP-N-Acetylglucosamine Enolpyruvyl Transferase, UDP-N-acetylenolpyruvylglucosamine reductase and Aspartate beta-semialdehyde dehydrogenase were identified as potential drug targets. Among these drug targets, UDP-N-acetylmu - ramate-L-alanine ligase (MurC) was chosen as potential therapeutic drug target. The homology modeling of MurC was performed using SWISS-MODEL and HHPred servers respectively. Subsequently, all the predicted models were evaluated using the SAVES server. The stereochemical parameters of all 3D models suggest that the best model was predicted by HHPred server. In order the refine the modeled structure, energy minimization was also performed using Deep View tool. The refined 3D structure was further validated by ProSA server. In future, the 3D structure of MurC in E. cloacae might be exploited for the discovery of novel inhibitors that could potentially inhibit this nosocomial pathogen.

Cloning, expression and characterization of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Wolbachia endosymbiont of human lymphatic filarial parasite Brugia malayi.

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (K m) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.

Identification of Novel Irreversible Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Haemophilus influenzae

Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 2.5.1.7) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-Nacetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC(50)) than fosfomycin, a wellknown inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor.

Identification of a novel fosfomycin-resistant UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from a soil metagenome

A soil metagenomic library was constructed and screened for clones that conferred fosfomycin resistance. A novel protein with 46% identity to UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Desulfuromonas acetoxidans DSM 684 (GenBank accession number: ZP_01311756) was identified. Multiple sequence alignment revealed that the novel protein was a natural MurA, in which an aspartic acid instead of a cysteine was located in the active site. An Asp120Cys mutant of Escherichia coli was constructed from the subclone through site-specific mutagenesis, and minimum inhibitory concentration of fosfomycin for the resistant subclone and its mutant were determined. These results showed that fosfomycin resistance was a result of the aspartic acid in the active site. Analysis of all existing MurA sequences revealed that MurAs with an active site aspartic acid that can confer fosfomycin resistance occur in ~14% of bacteria.

Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis

Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, only little is known about their complex biosynthesis. The genes encoding for proteins involved in the biosynthesis of the nucleoside moiety of nikkomycins are co-transcribed in the same operon, comprising the genes nikIJKLMNO. The gene product NikO was shown to belong to the family of enolpyruvyl transferases and to catalyze the transfer of an enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UMP. Here, we report activity and inhibition studies of the wild-type enzyme and the variants C130A and D342A. The x-ray crystal structure revealed differences between NikO and its homologs. Furthermore, our studies led to conclusions concerning substrate binding and preference as well as to conclusions about inhibition/alkylation by the antibiotic fosfomycin.

Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiological studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resolution of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry 2rl2) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron density for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clinically relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

Streptococcus mutans (S. mutans) is the primary etiological agent of human dental caries. A gene (SMU_1525) encoding MurA was identified in S. mutans UA159 (ATCC700610). The deduced amino-acid sequence has homology (51% identity) with E. coli MurA protein (AAC76221). In this study, we cloned S. mutans murA (SMU_1525) gene, expressed soluble recombinant MurA protein in E. coli BL21(DE3). The recombinant protein was purified by affinity chromatography. The molecular weight of expressed protein was about 45.6 kD. The activity of the protein was identified by high-pressure liquid chromatography (HPLC) and was shown to have UDP-N-acetylglucosamine enolpyruvyl transferase activity. Then one microtiter plate based colourimetric assay for S. mutans MurA activity was developed. The optimal temperature and pH of the purified enzyme were 37°C and 7.5, respectively. The Km for UDP-GlcNAc was 0.120 mM and for PEP was 0.086 mM. The Vmax for UDP-GlcNAc was 0.048 mM min-1 mg-1 and for PEP was 0.098 mM min-1 mg-1. The activity of S. mutans MurA was inhibited by the fosfomycin, known MurA inhibitor.

Comprehensive structural and functional characterization of Mycobacterium tuberculosis UDP-NAG enolpyruvyl transferase (Mtb-MurA) and prediction of its accurate binding affinities with inhibitors

Tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death in the world. One third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the etiologic agent of TB. The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. In this work, 3D structure model of Mtb-MurA enzyme has been developed for the first time by homology modeling and molecular dynamics simulation techniques. Multiple sequence alignment and 3D structure model provided the putative substrate binding pocket of Mtb-MurA with respect to E. coli MurA. This analysis was helpful in identifying the binding sites and molecular function of the MurA homologue. Molecular docking study was performed on this 3D structure model, using different classes of inhibitors like fosfomycin, cyclic disulfide analog RWJ-3981, pyrazolopyrimidine analog RWJ-110192, purine analog RWJ-140998, 5-sulfonoxy-anthranilic acid derivatives T6361, T6362 and the results showed that the 5-sulfonoxyanthranilic acid derivatives showed the best interaction compared to other inhibitors. We also designed new efficient analogs of T6361 and T6362 which showed even better interaction with Mtb-MurA than the parental 5-sulfonoxy-anthranilic acid derivatives. Further the comparative molecular electrostatic potential and cavity depth analysis of Mtb-MurA suggested several important differences in its substrate and inhibitor binding pocket. Such differences could be exploited in the future for designing a more specific inhibitor for Mtb-MurA enzyme.

UDP-N-acetylglucosamine enolpyruvyl transferase as a potential target for antibacterial chemotherapy: recent developments

The emergence of antibiotic resistance in bacterial pathogens has foxed the health organizations which are actively scrambling for solutions. The available data indicate an increased morbidity in infections often leading to mortality among patients where drug-resistant pathogens have negated the effect of the medicines. In the context of developing "novel bacterial inhibitors" for killing or arresting the growth of drug-resistant pathogens, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an enzyme that provides hope for the future. This enzyme catalyzes the first committed step in the biosynthesis of peptidoglycan, an integral and essential component of the bacterial cell wall. MurA enzyme is neither present nor required by mammals and shows poor homology with human proteins. Therefore, it is an ideal target for antibacterial chemotherapy. Till date, 18 structures of MurA (in native and ligand-bound forms) from different bacterial pathogens have been solved. In the last 2 years, eight structures of bacterial MurA have been submitted to the Protein Data Bank and many inhibitors discovered. The present review discusses the structural and functional features of MurA of bacterial pathogens along with the development of MurA-targeted inhibitors.

Benzothioxalone derivatives as novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferases (MurA and MurZ)

We sought to identify and characterize new inhibitors of MurA and MurZ, which are enzymes involved in the early stages of bacterial peptidoglycan synthesis. A library of ∼650 000 compounds was screened for inhibitors of Escherichia coli MurA in an endpoint assay measuring release of inorganic phosphate from phosphoenolpyruvate. Hits were validated by determining the concentrations required for 50% inhibition (IC(50)) of MurA from E. coli and MurA/MurZ from Staphylococcus aureus. The mode of action of selected inhibitors was explored by examining the reversibility of MurA inhibition, the binding of a radiolabelled inhibitor to MurA proteins and through docking studies. Inhibitors were further characterized by determining their antibacterial activity against E. coli and S. aureus. Benzothioxalone derivatives were identified that inhibited MurA from E. coli and MurA/MurZ from S. aureus with IC(50) values between 0.25 and 51 µM. Several inhibitors also exhibited activity against S. aureus with MICs in the range 4-128 mg/L. Inhibition of MurA was irreversible and a radiolabelled inhibitor from this compound class displayed stoichiometric binding to the enzyme, which was displaced by dithiothreitol. Binding was undetectable with a C115D mutant MurA protein. The results suggest a mode of action for the benzothioxalones that involves the formation of a disulfide bond with MurA/MurZ, via attack from an active site cysteine on the thioxalone ring carbonyl group, followed by ring opening to yield an S-acylated protein. The proposed covalent mode of action may prove useful in the design of new antibacterial agents.

UDP-N-acetylglucosamine enolpyruvyl transferase from Pseudomonas aeruginosa

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC50 for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.

Revealing fosfomycin primary effect on Staphylococcus aureus transcriptome: modulation of cell envelope biosynthesis and phosphoenolpyruvate induced starvation

BackgroundStaphylococcus aureus is a highly adaptable human pathogen and there is a constant search for effective antibiotics. Fosfomycin is a potent irreversible inhibitor of MurA, an enolpyruvyl transferase that uses phosphoenolpyruvate as substrate. The goal of this study was to identify the pathways and processes primarily affected by fosfomycin at the genome-wide transcriptome level to aid development of new drugs.ResultsS. aureus ATCC 29213 cells were treated with sub-MIC concentrations of fosfomycin and harvested at 10, 20 and 40 minutes after treatment. S. aureus GeneChip statistical data analysis was complemented by gene set enrichment analysis. A visualization tool for mapping gene expression data into biological pathways was developed in order to identify the metabolic processes affected by fosfomycin. We have shown that the number of significantly differentially expressed genes in treated cultures increased with time and with increasing fosfomycin concentration. The target pathway - peptidoglycan biosynthesis - was upregulated following fosfomycin treatment. Modulation of transport processes, cofactor biosynthesis, energy metabolism and nucleic acid biosynthesis was also observed.ConclusionsSeveral pathways and genes downregulated by fosfomycin have been identified, in contrast to previously described cell wall active antibiotics, and was explained by starvation response induced by phosphoenolpyruvate accumulation. Transcriptomic profiling, in combination with meta-analysis, has been shown to be a valuable tool in determining bacterial response to a specific antibiotic.

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 microg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

Inhibitory Mechanism of Novel Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Haemophilus influenzae

Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolphyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Heamophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations (IC50s) were found to range from 0.1 to 0.7 microM. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium at a concentration range of 1-2 microg/ml. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

Molecular modeling and bioinformatical analysis of the antibacterial target enzyme MurA from a drug design perspective

The enzyme MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first cytoplasmatic step in the synthesis of murein precursors. This function is of vital relevance for bacteria, and the enzyme therefore represents an important target protein for the development of novel antibacterial compounds. Several X-ray structures of liganded and un-liganded MurA have been published, which may be used for rational drug design. MurA, however, contains a highly flexible surface loop, which is involved in substrate and inhibitor binding. In the available X-ray structures, the conformation of this surface loop varies, depending on the presence or absence of ligands or substrate and probably also on the crystal packing. The uncertainty of the low-energy, or "resting state" conformation of this surface loop hampers the application of rational drug design to this class of enzymes. We have therefore performed an extensive molecular dynamics study of the enzyme in order to identify one or several low-energy conformers. The results indicate that, at least in some of the X-ray structures, the conformation of the flexible surface loop is influenced by crystallographic contacts. Furthermore, three partially helical foldamers of the surface loop are identified which may resemble the resting states of the enzyme or intermediate states that are "traversed" during the substrate binding process. Another, very important aspect for the development of novel antibacterial compounds is the inter- and intra-species variability of the target structure. We present a comparison of MurA sequences from 163 organisms which were analyzed under the aspects of enzyme mechanism, structure and drug design. The results allow us to identify the most promising binding sites for inhibitor interaction, which are present in MurA enzymes of most species and are expected to be insusceptible to resistance-inducing mutations.