Recombinant Treponema denticola UDP-N-acetylmuramyl-tripeptide synthetase (murE), partial

What is the fundamental role of UDP-N-acetylmuramyl-tripeptide synthetase (murE) in Treponema denticola?

MurE in T. denticola functions as the third amino acid-adding enzyme in the peptidoglycan biosynthesis pathway, specifically catalyzing the addition of meso-diaminopimelic acid (meso-DAP) to UDP-MurNAc-L-Ala-D-Glu . This step is essential for the creation of the peptidoglycan monomer that ultimately forms the bacterial cell wall structure. The enzyme acts as a ligase, forming an amide bond between the substrate and meso-DAP with concomitant ATP hydrolysis . As part of the Mur synthetase family, T. denticola MurE shares structural and functional features with other bacterial MurE enzymes while potentially exhibiting unique characteristics related to the oral treponeme's lifestyle and environment.

How does T. denticola murE contribute to bacterial survival and pathogenesis?

T. denticola murE contributes to bacterial survival by enabling peptidoglycan synthesis, which provides cell wall integrity and protection against osmotic stress . While T. denticola has been identified as an important cause of periodontal disease and potentially involved in extra-oral infections , the integrity of its cell wall is crucial for evading host immune responses. Although peptidoglycan has unique characteristics in spirochetes like T. denticola, the MurE enzyme remains essential for bacterial viability, making it a potential target for controlling infection. The cell wall components generated through the murE pathway may interact with immune surveillance mechanisms, affecting the bacterium's ability to colonize host tissues and resist clearance by phagocytic cells like macrophages .

What are the optimal conditions for expressing recombinant T. denticola murE in E. coli expression systems?

For optimal expression of recombinant T. denticola murE in E. coli systems, researchers should consider several critical parameters. Based on experiences with similar enzymes, expression should be performed in BL21(DE3) or similar strains designed for high-level protein expression. The murE gene should be cloned into a vector containing an inducible promoter system (such as T7) and ideally include a purification tag (6xHis or GST) to facilitate downstream purification. Induction conditions typically require optimization: IPTG concentrations ranging from 0.1-1.0 mM and induction at lower temperatures (16-25°C) for extended periods (16-20 hours) generally yield better results than standard conditions, helping to prevent inclusion body formation. It's crucial to note that unlike some other bacterial proteins, T. denticola proteins may require specific considerations due to their different codon usage patterns, potentially necessitating expression in Rosetta or CodonPlus strains that supply rare tRNAs.

What purification strategies yield the highest specific activity for recombinant T. denticola murE?

A multi-step purification process is recommended to achieve high specific activity of recombinant T. denticola murE. Following cell lysis (preferably using a French press or sonication in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors), an initial affinity chromatography step using Ni-NTA (for His-tagged constructs) or glutathione-Sepharose (for GST-tagged constructs) should be performed. For highest purity and activity, subsequent ion-exchange chromatography (typically Q-Sepharose) and size-exclusion chromatography are recommended. Throughout purification, it's critical to maintain the enzyme at 4°C and include stabilizing agents like glycerol (10-15%) and reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of critical cysteine residues. Activity assays should be performed after each purification step to track specific activity improvements. Researchers have observed that some recombinant MurE enzymes benefit from the inclusion of low concentrations of substrate analogs during purification to maintain the active site in an optimal conformation.

What are the established methodologies for measuring T. denticola murE enzymatic activity in vitro?

Several complementary approaches can be employed to measure T. denticola murE activity in vitro. The standard assay monitors the ATP-dependent ligation of meso-DAP to UDP-MurNAc-L-Ala-D-Glu . This can be quantified using:

• Coupled enzyme assays - where ADP production is linked to NADH oxidation through pyruvate kinase and lactate dehydrogenase, allowing continuous spectrophotometric monitoring at 340 nm.

• HPLC analysis - separating substrate and product based on their different retention times, typically using a reverse-phase C18 column with appropriate mobile phases.

• Radiolabeled substrate incorporation - using [14C]- or [3H]-labeled meso-DAP to measure product formation by scintillation counting after separation.

• Malachite green phosphate detection - measuring inorganic phosphate released during ATP hydrolysis.

The reaction buffer typically contains Tris-HCl (pH 8.0), MgCl2, KCl, and ATP. Optimal activity requires determining precise kinetic parameters (Km, Vmax) for each substrate (UDP-MurNAc-L-Ala-D-Glu, meso-DAP, and ATP) under varying conditions of pH, temperature, and ionic strength to establish the enzyme's biochemical properties.

How can researchers distinguish between the activity of T. denticola murE and other Mur synthetases in complex experimental systems?

Distinguishing T. denticola murE activity from other Mur synthetases requires multiple specific approaches. Researchers should employ substrate specificity analysis, as MurE specifically utilizes UDP-MurNAc-L-Ala-D-Glu and meso-DAP, while other Mur synthetases incorporate different amino acids . Selective inhibition patterns can also differentiate MurE, as each Mur enzyme exhibits unique sensitivity to specific inhibitors. Using selective antibodies against T. denticola murE in immunodetection or immunoprecipitation assays can isolate the enzyme from mixed samples.

For genetic approaches, selective gene knockout or silencing of murE can help attribute specific activity changes to this enzyme. Additionally, comparative kinetic analysis is valuable, as MurE and other Mur synthetases display characteristic kinetic parameters and metal ion dependencies. When possible, mass spectrometry analysis of reaction products provides definitive identification of the specific peptide additions catalyzed by murE versus other synthetases.

What structural features of T. denticola murE are critical for its catalytic function and specificity?

T. denticola murE, like other bacterial MurE ligases, likely contains three distinct domains that are critical for its function. The N-terminal domain typically binds UDP-MurNAc-dipeptide, the central domain binds ATP, and the C-terminal domain is responsible for meso-DAP binding . Key conserved motifs include the ATP-binding consensus sequence (P-loop) that coordinates the phosphate groups of ATP, and the characteristic amino acid binding pocket that confers specificity for meso-DAP over other amino acids .

The enzyme's catalytic mechanism involves a ternary complex formation, where ATP activation of the UDP-MurNAc-dipeptide C-terminal carboxyl group creates an acyl-phosphate intermediate, followed by nucleophilic attack by the amino group of meso-DAP . Critical catalytic residues likely include conserved acidic amino acids that coordinate the essential Mg2+ ions, and basic residues that stabilize transition states. While the precise structure of T. denticola murE has not been definitively characterized, comparative analysis with other bacterial MurE enzymes suggests these conserved features are present and essential for function.

How does the domain architecture of T. denticola murE compare with other bacterial murE enzymes?

The central domain containing the ATP-binding site likely demonstrates the highest conservation across bacterial species, featuring the classic ATP-binding consensus sequence (P-loop) characteristic of the ATP-grasp superfamily . The amino acid-binding C-terminal domain would show greater divergence, as it determines specificity for meso-DAP, yet maintains the key residues for substrate recognition.

T. denticola, as an oral pathogen with a distinctive cell envelope structure, may possess subtle modifications in its murE domains compared to model organisms like E. coli. These could include differences in surface charge distribution, altered loop regions affecting substrate access, or unique interdomain interactions affecting enzyme dynamics. These distinctive features may explain any observed differences in substrate specificity, catalytic efficiency, or inhibitor susceptibility between T. denticola murE and its counterparts in other bacterial species.

What approaches are most effective for generating site-directed mutations in T. denticola murE for structure-function studies?

For effective site-directed mutagenesis of T. denticola murE, researchers should employ a comprehensive approach beginning with in silico analysis. Homology modeling based on crystallized MurE structures from other bacteria should guide identification of catalytically important residues . For the experimental phase, QuikChange or Q5 site-directed mutagenesis are preferred methods when working with recombinant plasmids containing the murE gene.

When targeting multiple residues, overlap extension PCR or Gibson Assembly techniques may be more efficient. After transformation and sequence verification, expression and purification protocols should match those used for the wild-type enzyme to ensure valid comparisons. Comprehensive analysis of mutant enzymes should include:

• Steady-state kinetic analysis examining Km and kcat for all three substrates

• Binding studies using techniques like isothermal titration calorimetry

• Thermal stability assays to assess structural integrity

• Product analysis by HPLC or mass spectrometry

Interpreting results requires careful consideration of both direct catalytic effects and potential conformational changes. Circular dichroism spectroscopy can provide valuable information about any significant structural alterations resulting from mutations.

How can conditional knockout or depletion of murE in T. denticola be achieved to study its essentiality for cellular viability?

Creating conditional knockouts or depletion systems for murE in T. denticola requires specialized genetic tools adapted for this challenging spirochete. An inducible expression system should be established first, where the native murE gene is deleted and replaced with a copy under control of an inducible promoter. The tetracycline-inducible system has been adapted for use in some spirochetes and could potentially work in T. denticola, allowing gradual depletion of MurE by withdrawal of the inducer.

Alternatively, a complementation approach could utilize a temperature-sensitive plasmid carrying murE that replicates at permissive temperatures but is lost at non-permissive temperatures. For antisense RNA-based depletion, researchers should design antisense constructs targeting the murE mRNA and place them under an inducible promoter to titrate expression levels.

After establishing these systems, researchers should monitor:

• Growth curves at various depletion levels

• Cell morphology changes using microscopy techniques

• Peptidoglycan content and composition

• Cell integrity under osmotic stress conditions

• Susceptibility to cell wall-targeting antibiotics

These approaches would provide crucial evidence regarding the degree of essentiality of murE for T. denticola viability and potential compensatory mechanisms that might exist in this oral pathogen.

How does T. denticola murE differ from other oral spirochete murE enzymes in terms of sequence conservation and substrate specificity?

T. denticola murE shows notable sequence conservation patterns when compared with murE enzymes from other oral spirochetes, reflecting both their shared evolutionary history and specialized habitat adaptations. While the ATP-binding domain displays high conservation across all bacterial murE enzymes (typically >70% similarity), the substrate-binding domains of oral spirochete murE enzymes show more divergence.

These differences may manifest in slight variations in substrate specificity or catalytic efficiency. While all characterized bacterial MurE enzymes incorporate meso-DAP at position 3 of the peptidoglycan stem peptide in gram-negative bacteria, T. denticola and related oral spirochetes may show differences in their affinity constants (Km) or catalytic rates (kcat) with the UDP-MurNAc-L-Ala-D-Glu substrate. These kinetic differences likely reflect adaptations to the specialized oral niche and particular cell wall requirements of different spirochete species.

What insights can comparative genomics provide about the evolution of the murE gene in T. denticola and other spirochetes?

The gene neighborhood analysis of murE in T. denticola reveals conservation of the dcw (division and cell wall) gene cluster organization found in many bacteria, though some rearrangements may be observed compared to model organisms. While the core catalytic regions show strong purifying selection (dN/dS ratios <1), certain surface-exposed loops may display higher rates of sequence variation, potentially reflecting adaptation to specific environmental conditions or interactions with other cellular components.

What methodologies are most appropriate for screening potential inhibitors of T. denticola murE?

A comprehensive approach to screening potential inhibitors of T. denticola murE should employ multiple complementary methodologies. High-throughput primary screening can utilize a coupled enzymatic assay that monitors ADP production through linked reactions with pyruvate kinase and lactate dehydrogenase, measuring NADH oxidation at 340 nm. This approach allows rapid screening of large compound libraries in 384-well format.

For confirmation and detailed characterization of hits, researchers should employ orthogonal assays including:

• Direct product analysis by HPLC or LC-MS to confirm specific inhibition of product formation

• Thermal shift assays (differential scanning fluorimetry) to identify compounds that bind to and stabilize murE

• Surface plasmon resonance or isothermal titration calorimetry to determine binding constants and thermodynamic parameters

• Enzymatic assays with varying substrate concentrations to determine the mechanism of inhibition (competitive, non-competitive, or uncompetitive)

To assess selectivity, parallel testing against human ATP-utilizing enzymes is essential. Compounds showing promising in vitro activity should be evaluated in cellular assays to determine their ability to penetrate the outer membrane of T. denticola and affect peptidoglycan synthesis. Structure-activity relationship studies of effective inhibitors can guide medicinal chemistry optimization for improved potency and selectivity.

How can structural information about T. denticola murE be leveraged for structure-based drug design approaches?

Without a crystal structure specifically for T. denticola murE, researchers must initially rely on homology modeling based on solved structures of MurE from other bacterial species . This approach should incorporate sequence alignment data and molecular dynamics simulations to refine the model. Key structural features to focus on include the ATP-binding pocket, the UDP-MurNAc-dipeptide binding site, and the meso-DAP binding region.

For structure-based drug design, multiple computational approaches should be employed:

• Molecular docking studies to virtually screen compound libraries against the modeled active site

• Pharmacophore modeling based on known ligands and substrate interactions

• Fragment-based design targeting specific sub-pockets within the active site

• Molecular dynamics simulations to identify transient binding pockets and assess ligand stability

Drug design strategies should exploit unique features of T. denticola murE compared to human enzymes, focusing on selective inhibition. Rational design approaches might include:

• ATP-competitive inhibitors with modifications to enhance selectivity

• Transition state analogs mimicking the phosphorylated intermediate

• Allosteric inhibitors targeting regulatory sites unique to bacterial murE enzymes

• Covalent inhibitors targeting non-conserved cysteine residues

As lead compounds are identified, medicinal chemistry optimization should focus on improving not only binding affinity but also physicochemical properties to enhance penetration through the T. denticola outer membrane, a critical consideration for effective antimicrobial activity against this oral pathogen.

What are the most common technical challenges in working with recombinant T. denticola murE and how can they be addressed?

Researchers working with recombinant T. denticola murE frequently encounter several technical challenges that require specific troubleshooting approaches:

• Low soluble expression levels: Many researchers observe inclusion body formation when expressing spirochete proteins in E. coli. This can be addressed by:

  • Reducing induction temperature to 16-18°C

  • Using specialized expression strains (Rosetta, Arctic Express)

  • Adding solubility-enhancing fusion tags (SUMO, MBP)

  • Exploring cell-free expression systems when standard approaches fail

• Enzyme instability during purification: T. denticola murE may show decreased activity during purification steps. Stability can be improved by:

  • Including glycerol (10-20%) in all buffers

  • Adding reducing agents (5 mM DTT or 10 mM β-mercaptoethanol)

  • Maintaining strict temperature control (4°C)

  • Including substrate analogs or product mimics at low concentrations

• Inconsistent enzymatic activity: Batch-to-batch variation in activity assays is common and can be minimized by:

  • Carefully controlling enzyme:substrate ratios

  • Including positive controls with each assay

  • Validating substrate quality before each experiment

  • Standardizing enzyme storage conditions (small aliquots, -80°C)

• Difficulty distinguishing murE activity from contaminating enzymes: This can be addressed through:

  • Additional purification steps (ion exchange, size exclusion)

  • Specific activity measurements after each purification step

  • Control experiments with heat-inactivated enzyme

  • Western blot verification of purified protein identity

These approaches have significantly improved success rates in working with this challenging enzyme from an oral pathogen with specialized growth requirements.

How can researchers optimize assay conditions for detecting low levels of T. denticola murE activity in complex biological samples?

Detecting low levels of T. denticola murE activity in complex biological samples requires careful optimization of several experimental parameters. Researchers should first consider sample preparation techniques that enrich for murE activity while minimizing interfering components. This may include:

• Gentle cell lysis methods that preserve enzyme activity

• Sub-fractionation techniques to separate cytoplasmic components

• Ammonium sulfate precipitation to concentrate enzymatic activity

• Initial cleanup using ion exchange or affinity chromatography

For the activity assay itself, several modifications can enhance sensitivity:

• Extended incubation times (up to 16 hours) at lower temperatures (25-30°C) to allow product accumulation while minimizing degradation

• Increased substrate concentrations (2-5× Km) to drive the reaction toward product formation

• Addition of stabilizing agents like BSA (0.1-1 mg/mL) and glycerol (10%)

• Inclusion of phosphatase inhibitors to prevent degradation of the acyl-phosphate intermediate

Detection methods should be optimized for maximum sensitivity:

• Radiometric assays using high specific activity [14C]- or [3H]-labeled meso-DAP

• LC-MS/MS detection with selected reaction monitoring for specific product fragments

• Coupled enzyme systems with signal amplification through cycling reactions

• Fluorescent or bioluminescent readouts for enhanced sensitivity

Control experiments are critical and should include:

• Spiking samples with known amounts of recombinant enzyme to establish recovery rates

• Parallel assays with specific murE inhibitors to confirm signal specificity

• Heat-inactivated samples to establish true background levels

These optimizations can improve detection limits by 10-100 fold compared to standard protocols, enabling meaningful measurements even in samples with low bacterial loads.

How might cryo-EM techniques advance our understanding of T. denticola murE structure and function beyond what X-ray crystallography has revealed?

Cryo-electron microscopy (cryo-EM) offers several distinct advantages for elucidating T. denticola murE structure and function. Unlike X-ray crystallography, which has been challenging for many Mur enzymes due to conformational flexibility, cryo-EM can capture multiple conformational states simultaneously in a near-native environment. This is particularly valuable for understanding the substantial domain movements that likely occur during the MurE catalytic cycle as it transitions between open and closed conformations during substrate binding and product release.

Cryo-EM could specifically reveal:

• The complete conformational landscape of T. denticola murE, including intermediates not captured in crystal structures

• Details of protein dynamics during catalysis, particularly the orientation of domains relative to each other

• Potential oligomerization states that might exist under physiological conditions

• Interactions with other components of the peptidoglycan biosynthetic machinery in more complex preparations

Recent advances in cryo-EM technology, particularly the development of micro-electron diffraction (microED) for smaller proteins and the use of Volta phase plates to enhance contrast, make it increasingly feasible to achieve near-atomic resolution for enzymes the size of murE (~55 kDa). This approach could overcome the persistent challenges of obtaining diffraction-quality crystals and provide novel insights into the structural basis of T. denticola murE function, potentially revealing unique features that could be exploited for selective inhibitor design.

What are the emerging approaches for studying T. denticola murE interactions with other components of the peptidoglycan biosynthetic machinery?

Emerging approaches for studying T. denticola murE interactions with other peptidoglycan biosynthetic components increasingly rely on integrative methodologies that capture both physical interactions and functional relationships. Proximity-dependent labeling techniques, such as BioID or APEX2, allow identification of proteins that come into close contact with murE in the native cellular environment. These approaches can be particularly valuable for mapping the dynamic interactome of murE throughout the bacterial cell cycle.

Advanced microscopy techniques, including single-molecule localization microscopy (PALM/STORM) coupled with multi-color labeling, can visualize the co-localization of murE with other peptidoglycan synthesis enzymes at nanometer resolution. This can reveal spatial and temporal patterns in the assembly of the peptidoglycan biosynthetic machinery that conventional co-immunoprecipitation methods might miss.

Synthetic biology approaches are also emerging as powerful tools, where minimally engineered peptidoglycan synthesis systems are reconstituted in vitro from purified components. Such systems allow precise control over component concentrations and conditions, enabling detailed kinetic analysis of how murE activity is modulated by other enzymes in the pathway.

Computational approaches, including molecular docking and molecular dynamics simulations of multi-protein complexes, can provide atomic-level insights into potential interaction interfaces. These predictions can guide targeted mutagenesis of surface residues to disrupt specific protein-protein interactions and assess their functional significance.