Mur Ligases Research Articles

Antibiotics and Antibiotic Resistance-Mur Ligases as an Antibacterial Target.

The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic pathway represents opportunities for the development of novel antibacterial agents. Among the enzymes involved, Mur ligases, described herein, and especially the amide ligases MurC-F are key targets for the discovery of multi-inhibitors, as they share common active sites and structural features.

Computational and Biophysical Approaches to Identify Cell Wall-Associated Modulators in Salmonella enterica serovar Typhi.

An increase in the number of antibiotic-resistant bacterial pathogens, in recent times, has posed a great challenge for treating the affected patients. This has paved the way for the development and design of antibiotics against the previously less explored newer targets. Among these, peptidoglycan (PG) biosynthesis serves as a promising target for the design and development of novel drugs. The peptidoglycan cell wall synthesis in bacteria is essential for its viability. The enzyme class, Mur ligases, plays a key role in PG biosynthesis. Therefore, compounds with the ability to inhibit these enzymes (Mur ligase) can serve as potential candidates for developing small modulators. The enzyme, UDP-N-acetyl pyruvyl-glucosamine reductase(MurB), is essential for PG biosynthesis, a crucial part of the bacterial cell wall. The development of novel drugs to treat infections may thus focus on inhibiting MurB function. Understanding the mechanism of action of Mur B is central to developing efficient inhibitors. For the treatment of S. typhi infections, it is also critical to find therapeutic drugs that specifically target MurB. The enzyme Mur B from Salmonella enterica serovar Typhi (stMurB) was expressed and purified for biophysical characterization to gauge the molecular interactions and estimate thermodynamic stability, for determining attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism (CD) and validated by performing differential scanning calorimetry (DSC). An in silico virtual screening of various natural inhibitors was conducted with modelled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) obtained from in silico screening were validated for complex stability through molecular dynamics (MD) simulation. Further, fluorescence binding studies were undertaken for the selected natural inhibitors with stMurB alone and with its NADPH-bound form. The natural inhibitors, scopoletin and berberine, displayed lesser binding to stMurB compared to quercetin. Also, a stronger binding affinity was exhibited between quercetin and stMurB compared to NADPH and stMurB. Based on the above two findings, quercetin can be developed as an inhibitor of stMurB enzyme.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

The crystal structure of Mycobacterium thermoresistibile MurE ligase reveals the binding mode of the substrate m-diaminopimelate

The cytoplasmatic biosynthesis of the stem peptide from the peptidoglycan in bacteria involves six steps, which have the role of three ATP-dependent Mur ligases that incorporate three consecutive amino acids to a substrate precursor. MurE is the last Mur ligase to incorporate a free amino acid. Although the structure of MurE from Mycobacterium tuberculosis (MtbMurE) was determined at 3.0 Å, the binding mode of meso-Diaminopimelate (m-DAP) and the effect of substrate absence is unknown. Herein, we show the structure of MurE from M. thermoresistibile (MthMurE) in complex with ADP and m-DAP at 1.4 Å resolution. The analysis of the structure indicates key conformational changes that the substrate UDP-MurNAc-L-Ala-D-Glu (UAG) and the free amino acid m-DAP cause on the MthMurE conformation. We observed several movements of domains or loop regions that displace their position in order to perform enzymatic catalysis. Since MthMurE has a high similarity to MtbMurE, this enzyme could also guide strategies for structure-based antimicrobial discovery to fight against tuberculosis or other mycobacterial infections.

Identification of natural small molecule modulators of MurB from Salmonella enterica serovar Typhi Ty2 strain using computational and biophysical approaches.

The increase of antibiotic-resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in PG biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and hence, form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP-N-acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism and validated through differential scanning calorimetry experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical-induced denaturation of stMurB. In the search for natural compound-based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through molecular dynamics simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme.

Thiazolidin-2,4-Dione Scaffold: An Insight into Recent Advances as Antimicrobial, Antioxidant, and Hypoglycemic Agents

Heterocyclic compounds containing nitrogen and sulfur, especially those in the thiazole family, have generated special interest in terms of their synthetic chemistry, which is attributable to their ubiquitous existence in pharmacologically dynamic natural products and also as overwhelmingly powerful agrochemicals and pharmaceuticals. The thiazolidin-2,4-dione (TZD) moiety plays a central role in the biological functioning of several essential molecules. The availability of substitutions at the third and fifth positions of the Thiazolidin-2,4-dione (TZD) scaffold makes it a highly utilized and versatile moiety that exhibits a wide range of biological activities. TZD analogues exhibit their hypoglycemic activity by improving insulin resistance through PPAR-γ receptor activation, their antimicrobial action by inhibiting cytoplasmic Mur ligases, and their antioxidant action by scavenging reactive oxygen species (ROS). In this manuscript, an effort has been made to review the research on TZD derivatives as potential antimicrobial, antioxidant, and antihyperglycemic agents from the period from 2010 to the present date, along with their molecular mechanisms and the information on patents granted to TZD analogues.

Synthesis and Antibacterial Evaluation of Rhodanine and Its Related Heterocyclic Compounds against S. aureus and A. baumannii.

Antimicrobial resistance is a serious challenge to modern medicine. Besides imposing high financial burden, multidrug resistant infections are directly responsible for high morbidity and mortality. Even though a number of antibiotics are currently available to treat infections caused by ESKAPE organisms, more and more bacterial strains are becoming resistant to these drugs. Prevailing circumstances pose an urgent unmet need for the development of newer antimicrobials to treat the infections caused by MDR organisms. Rhodanine and structurally related 5-membered heterocycles possess wide range of pharmacological activities. A number of these derivatives have shown good to potent inhibition against various microorganisms. They are reported to alter the function of DNA gyrase B, metallo-β-lactamases, penicillin binding protein (PBP), Mur ligases, RNA polymerase, Enoyl ACP reductases, 1-deoxy-d-xylulose-5-phosphate reductoisomerase. etc which are vital in bacterial growth, survival and replication. In this study, we have generated a library of Rhodanine and related 5 membered heterocyclic derivatives and screened them against a panel of pathogens. Among all the compounds, 2a-i, 3a-b, 3g, 4, 6b-c, 6e, 6g, 12a-b and 14b-c have demonstrated good to moderate inhibition against S. aureus (MIC 0.125-8 μg/mL). Further, compound 17b demonstrated moderate activity against A. baumannii (MIC 8 μg/mL). In addition, compounds 2a, 2e, 4, 6c, 6g and 14b have shown good to mild inhibition against MDR S. aureus including VRSA (MIC 0.5-16 μg/mL) with good selectivity index 20-1600. In addition, compound 2e inhibited the growth gradually after 6 h in time kill kinetic studies and not antagonized with the tested FDA approved drugs.

Mur ligases inhibitors with azastilbene scaffold: Expanding the structure–activity relationship

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC–MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure–activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.

Design and Synthesis of Various 5'-Deoxy-5'-(4-Substituted-1,2,3-Triazol-1-yl)-Uridine Analogues as Inhibitors of Mycobacterium tuberculosis Mur Ligases.

The synthesis of hitherto unknown 5′-deoxy-5′-(4-substituted-1,2,3-triazol-1-yl)-uridine and its evaluation, through an one-pot screening assay, against MurA-F enzymes involved in Mycobacterium tuberculosis (Mtb), are described. Starting from UDP-N-acetylmuramic acid (UDP-MurNAc), the natural substrate involved in the peptidoglycan biosynthesis, our strategy was to substitute the diphosphate group of UDP-MurNAc by a 1,2,3-triazolo spacer under copper-catalyzed azide-alkyne cycloaddition conditions. The structure-activity relationship was discussed and among the 23 novel compounds developed, N-acetylglucosamine analogues 11c and 11e emerged as the best inhibitors against the Mtb MurA-F enzymes reconstruction pathway with an inhibitory effect of 56% and 50%, respectively, at 100 μM. Both compounds are selective inhibitors of Mtb MurE, the molecular docking and molecular dynamic simulation suggesting that 11c and 11e are occupying the active site of Mtb MurE ligase.

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases, MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

Complex Formation between Mur Enzymes from Streptococcus pneumoniae.

Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

Cell wall peptidoglycan in Mycobacterium tuberculosis: An Achilles' heel for the TB-causing pathogen.

ABSTRACTTuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE–MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.

Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32–368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.

Reaching toward underexplored targets in antibacterial drug design.

The increase of antimicrobial resistance necessitates the renewal and strong research involvement in antibacterial drug design. In the following work, we comment on the key approaches used in development of new antibacterials, focusing on intracellular therapeutic targets that have been so far mostly underexplored: the enzymes of the Mur pathway MurA to MurF. We identify common obstacles observed during research on MurA, MurB, and Mur ligases inhibitors and their development into potential antibacterial compounds, and discern several approaches and solutions to tackle the whole-cell activity of designed compounds. Furthermore, we consolidate recent literature reports and encourage the further research on Mur enzymes.

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, we present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.

A computational approach using mathematical modeling to assess the peptidoglycan biosynthesis of Clostridium botulinum ATCC 3502 for potential drug targets

C.botulinum ATCC 3502 is an obligate rod-shaped spore forming anaerobe causing food poisoning cases worldwide. The increased burden of sporadic hospital outbreaks reflects the pandemicity caused by this pathogen. Several attempts to control the disease surveillance, emergence of antibiotic resistant strains plagued the defense provided by the drugs. Peptidoglycan biosynthesis of C.botulinum ATCC 3502 is targeted to identify potential drug targets due to presence of no functional homolog in host (Homo sapiens). The role of peptidoglycan is to provide strength, fragility and protection to bacterial cells; therefore, it is considered as an attractive target for drug target identification. This study involves systematic exploration of targeted pathway by performing metabolic pathways analysis in two phases (1) mathematical modeling (2) elementary mode analysis (EM). Performing stoichiometric and kinetic modeling discerns the steady state conditions of the system to scrutinize elementary nodes with well-defined objective function. The study identifies Mur ligase enzymes (murA, murB, murC, murD), D-alanine-D-alanine ligase and glutamate racemase potential therapeutic targets for drug discovery. Further, the quantitative analysis characterized their potential in causing the pathogenicity. Physicochemical characterization and subcellular localization analysis assist in understanding biological activity of identified elementary nodes under different environmental conditions.

Docking study with biological validation on bacterial enzyme MurD

Abstract Approved antibiotics currently target only a small number of bacterial enzymes, despite increasing rates of antibiotic-resistant microbes. New chemical entities, especially with a novel mode of action, are ferociously being sought. To determine whether the ubiquitous bacterial enzyme Murein ligase D (MurD) could be inhibited in an alternative way to existing inhibitors, we performed molecular docking on a distinct site of the protein with potential new inhibitory properties. The compounds ranked highest by the docking program were acquired and screened for in vitro activity on isolated MurD. We found that the screened compounds exerted only marginal enzyme inhibitory activity, leading us to the conclusion that our hypothesis on a potential new binding site of MurD was too optimistic. We supplement this article with the names and structures of the screened molecules, to further assist is the search for novel antibiotic binding sites on Mur ligases.