Recombinant Protochlamydia amoebophila UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA)

What is the Function of MurA in Bacterial Peptidoglycan Synthesis?

MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) catalyzes the first committed step in peptidoglycan biosynthesis, which is essential for bacterial cell wall formation. The enzyme transfers an enolpyruvyl group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDPAG) to form UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine .

This reaction occurs in the cytoplasm as part of a series of steps where six enzymes (MurA to MurF) catalyze the formation of the soluble precursor UDP-MurNAc-pentapeptide . The pyruvate moiety provided by this reaction serves as the linker that bridges the glycan and peptide portions of peptidoglycan .

In obligate intracellular bacteria like Protochlamydia amoebophila, the peptidoglycan biosynthesis pathway appears to be functional despite these organisms living within the isotonic environment of eukaryotic cells, suggesting selective pressure to maintain this pathway even when osmotic protection is provided by the host cell .

How Can Recombinant P. amoebophila MurA Be Cloned and Expressed?

Successful cloning and expression of P. amoebophila MurA requires several methodological considerations:

Cloning Procedure:

• Amplify the murA gene using PCR with primers containing appropriate restriction sites (e.g., BamHI and XhoI as used for Wolbachia MurA)

• Subclone the amplified product initially into a T/A cloning vector

• Transfer to a bacterial expression vector (e.g., pET28a) using the engineered restriction sites

• Transform into an appropriate E. coli expression strain (e.g., Rosetta(DE3)pLysS)

Expression Optimization:

• Induce expression at lower temperatures (20-24°C) to enhance soluble protein production

• Use reduced IPTG concentrations (0.2-0.5 mM) to minimize inclusion body formation

• Extend induction time (20-22 hours) at lower temperature to increase yield of soluble protein

• Include appropriate antibiotics for plasmid maintenance

Purification Strategy:

• Use immobilized metal affinity chromatography with His-tagged constructs

• Optimize buffer conditions by varying pH and salt concentration

• Expected yield: approximately 0.3 mg of purified recombinant protein per liter of culture

This approach has been successful for the related Wolbachia MurA and can be adapted for P. amoebophila MurA with appropriate modifications to address specific solubility or expression challenges.

What Methods Are Used to Measure MurA Enzymatic Activity In Vitro?

Several robust assays have been developed to measure MurA enzymatic activity:

1. Malachite Green Assay:

• Measures the release of inorganic phosphate using malachite green and sodium molybdate

• The phosphate-reagent complex forms a green solution quantifiable spectrophotometrically

• Advantages: high sensitivity, suitable for high-throughput screening

2. D-Amino Acid Oxidase Coupled Assay:

• Used for detecting alanine racemase activity, which can be a side activity of some enzymes

• D-Ala produced is converted to pyruvate by D-amino acid oxidase (DAAO)

• Pyruvate is then colorimetrically quantified

3. Direct Product Detection:

• LC-MS or HPLC-based methods to directly measure the formation of UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine

• Enables detailed kinetic analysis and mechanism studies

Typical Reaction Conditions:

• Buffer: 50 mM HEPES, pH 7.5

• Temperature: 37°C (optimal for most MurA enzymes)

• Substrate concentrations: UDP-N-acetylglucosamine (0.01-1 mM) and phosphoenolpyruvate (0.01-1 mM)

• Reaction time: 15-30 minutes

For P. amoebophila MurA specifically, the expected kinetic parameters would likely be similar to related MurA enzymes, with Km values in the micromolar range for both substrates (as observed with Wolbachia MurA: Km = 0.03149 mM for UDP-N-acetylglucosamine and 0.009198 mM for phosphoenolpyruvate) .

How Does Fosfomycin Inhibit MurA and What Resistance Mechanisms Exist?

Fosfomycin is a clinically important antibiotic that specifically targets MurA through a well-characterized mechanism:

Inhibition Mechanism:

• Fosfomycin acts as a structural analog of phosphoenolpyruvate (PEP)

• It covalently modifies the active site cysteine residue (Cys115 in E. coli MurA)

• This irreversible reaction prevents substrate binding and catalysis

Known Resistance Mechanisms:

• Active Site Mutations:

  • Substitution of the active cysteine with aspartate (C115D) confers fosfomycin resistance

  • This mutation has been observed in Chlamydia trachomatis and Mycobacterium tuberculosis

  • It preserves enzymatic activity while preventing fosfomycin binding

• MurA Escape Mutations:

  • Mutations outside the active site (e.g., N197D and S262L identified in Listeria monocytogenes)

  • These mutations prevent normal proteolytic degradation of MurA

  • Result in increased MurA stability and levels, enhancing cell wall biosynthesis

• Alternative Binding Sites:

  • Novel inhibitors like pyrrolidinediones can target MurA at sites distinct from the fosfomycin binding site

  • These compounds inhibit both wild-type and fosfomycin-resistant MurA (C115D mutant)

The sensitivity of P. amoebophila MurA to fosfomycin would depend on the conservation of the active site cysteine residue, which appears to be maintained in most MurA enzymes from Chlamydiales including Wolbachia MurA that shows approximately 2-fold inhibition in the presence of fosfomycin .

What Structural Features Are Important for P. amoebophila MurA Function?

Several key structural features are critical for MurA enzymatic function based on structural and biochemical studies of related MurA enzymes:

Domain Architecture:

• MurA proteins comprise two globular domains connected by a double-stranded linker

• The UDP-GlcNAc binding site and catalytic center are located at the interface between these domains

• A significant conformational change occurs upon substrate binding, bringing the domains together

Critical Active Site Residues:

• Lys22: Forms covalent adducts with PEP and fosfomycin

• Cys115: Target for fosfomycin binding and involved in catalysis

• Asp305: Involved in product release and final deprotonation step

• Asp369 and Leu370: Facilitate fosfomycin interaction

Structural Dynamics:

• MurA undergoes a transition from an "open" to a "closed" conformation upon substrate binding

• This conformational change is essential for catalysis and involves a 20° domain closure

• In some bacteria, MurA exists in a "dormant" complex with UDP-MurNAc (UNAM) and covalently bound PEP

Sequence Conservation:
Sequence analysis suggests that P. amoebophila MurA would contain these conserved features. The Chlamydiales MurA enzymes are expected to maintain the key catalytic residues while potentially having unique structural features that reflect their adaptation to an intracellular lifestyle and specific regulatory mechanisms .

How Do MurA Escape Mutations Affect Peptidoglycan Biosynthesis Regulation?

MurA escape mutations have profound effects on peptidoglycan biosynthesis regulation, as demonstrated by studies in Listeria monocytogenes:

Mechanism of Escape Mutations:

• Mutations like N197D and S262L in L. monocytogenes MurA prevent protein-protein interactions with ReoM

• This disruption prevents MurA from being targeted for proteolytic degradation by the ClpCP protease

• The result is increased stability and elevated levels of MurA enzyme

Impact on Protein Interactions:

• Wild-type MurA interacts with ReoM in bacterial two-hybrid systems and pull-down assays

• MurA variants N197D and S262L showed significantly reduced interaction with ReoM (13±6% and 19±11% of wild-type levels)

• These mutations are located in surface-exposed helical regions outside the active site

Effects on Peptidoglycan Regulation:

• Increased MurA Stability:

  • MurA N197D and S262L variants showed delayed degradation compared to wild-type

  • N197D and S262L MurA levels were 1.8-fold and 2.1-fold higher than wild-type, respectively

• Rescue of Growth Defects:

  • Lower IPTG concentrations (0.05 mM vs 0.5 mM required for wild-type) were sufficient for MurA escape mutants to rescue growth defects in a ΔgpsB strain

  • This indicates enhanced functionality at lower expression levels

• Enhanced Cephalosporin Resistance:

  • Escape mutations enhanced intrinsic cephalosporin resistance

  • This effect required functional RodA3/PBP B3 transglycosylase/transpeptidase pair

These findings demonstrate that MurA escape mutations effectively uncouple peptidoglycan biosynthesis from normal regulatory constraints, highlighting the importance of proteolytic control in this pathway.

What Are the Implications of Targeting MurA in Obligate Intracellular Bacteria for Antimicrobial Development?

Targeting MurA in obligate intracellular bacteria presents unique opportunities and challenges for antimicrobial development:

Strategic Advantages:

• Essential Target:

  • MurA catalyzes the first committed step in peptidoglycan biosynthesis

  • Genomic evidence indicates this pathway is conserved across Chlamydiales despite their intracellular lifestyle

  • Deletion or inactivation of MurA is lethal in multiple bacterial species

• Selective Toxicity:

  • No eukaryotic homologs exist, minimizing host toxicity concerns

  • Inhibition affects bacterial cell integrity without directly harming host cells

Challenges and Considerations:

• Metabolic State Dependency:

  • Efficacy may vary depending on the developmental stage (elementary bodies vs. reticulate bodies)

  • P. amoebophila elementary bodies maintain metabolic activity, including glucose metabolism and anabolic reactions

• Resistance Development:

  • Fosfomycin resistance can occur through point mutations in the active site

  • Designing inhibitors that maintain efficacy against resistant mutants is crucial

• Intracellular Penetration:

  • Compounds must cross host cell membranes to reach intracellular bacteria

  • They must also penetrate bacterial membranes, which may have unique composition in obligate intracellular species

Novel Approaches:

• Multi-target Inhibitors:

  • Pyrrolidinedione-based MurA inhibitors show activity against both wild-type and fosfomycin-resistant MurA

  • Compounds that target multiple enzymes in the peptidoglycan pathway may reduce resistance development

• Host-Symbiont Interactions:

  • In Wolbachia (an endosymbiont of filarial nematodes), targeting MurA affects viability of both the bacterium and the host nematode

  • This approach could be valuable for diseases like lymphatic filariasis

• Developmental Stage Targeting:

  • Research shows P. amoebophila elementary bodies require D-glucose for maintaining infectivity

  • Combining MurA inhibitors with glucose metabolism disruptors might enhance efficacy

How is MurA Expression Regulated in Chlamydial Species?

Regulation of MurA in chlamydial species involves complex mechanisms reflecting their unique developmental cycle:

Developmental Stage-Specific Expression:

• In Chlamydiaceae, peptidoglycan synthesis genes (including MurA) are primarily expressed during reticulate body development and division

• Expression patterns correlate with the metabolic needs during different life cycle stages

Serine/Threonine Protein Kinase-Mediated Regulation:

• In Listeria monocytogenes, MurA is regulated by the serine/threonine protein kinase PrkA

• This regulation occurs via the small cytosolic protein ReoM, which when phosphorylated prevents MurA degradation by the ClpCP protease

• Similar kinase-based regulatory systems may exist in Chlamydiales

Proteolytic Control:

• MurA stability is actively regulated through proteolytic degradation

• In L. monocytogenes, the ClpCP protease system targets MurA for degradation

• ReoM and ReoY act as adaptor-like proteins facilitating this degradation

• This regulatory mechanism likely represents a conserved approach to controlling peptidoglycan synthesis

Metabolic Regulation:

• P. amoebophila elementary bodies maintain metabolic activity including glucose catabolism

• The pentose phosphate pathway was identified as the major route of D-glucose catabolism

• This metabolic activity is essential for maintaining infectivity

• These metabolic pathways may indirectly regulate MurA activity by controlling substrate availability

Cross-talk with Cell Division Machinery:

• In L. monocytogenes, mutations in MurA can suppress growth defects in strains lacking the cell division protein GpsB

• This suggests coordination between peptidoglycan synthesis and cell division processes

Understanding these regulatory mechanisms provides insights into potential intervention points for antimicrobial development and explains how obligate intracellular bacteria adjust peptidoglycan synthesis to their intracellular lifestyle.

What Role Do Post-Translational Modifications Play in MurA Function?

Post-translational modifications significantly influence MurA function through several mechanisms:

Covalent Substrate Interactions:

• MurA can form a covalent adduct with its substrate phosphoenolpyruvate (PEP)

• This phospholactoyl adduct involves active site Cys115 and has important functional consequences

• X-ray crystallography studies have captured this adduct in various reaction states

Functional Consequences of PEP Adduct Formation:

• "Dormant" Complex Formation:

  • Cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM)

  • PEP is covalently attached to Cys115 in this complex

  • This complex serves as a regulatory mechanism for peptidoglycan biosynthesis

• Reaction Priming:

  • The covalent reaction with PEP appears to prime the PEP molecule for instantaneous reaction with UDP-N-acetylglucosamine

  • This enables efficient catalysis when peptidoglycan synthesis is activated

Phosphorylation-Dependent Regulation:

• While direct phosphorylation of MurA has not been widely reported, phosphorylation of regulatory proteins affects MurA stability

• In L. monocytogenes, phosphorylation of ReoM by the serine/threonine kinase PrkA prevents MurA degradation

• Conversely, dephosphorylated ReoM promotes MurA degradation by the ClpCP protease

Implications for Structural Studies:

• The discovery of the MurA-PEP adduct revealed that many previously published crystal structures of MurA from various organisms were misinterpreted

• These structures actually contained UNAM and covalently bound PEP

• This finding explains challenges in developing effective MurA inhibitors, as the enzyme's predominant cellular state differs from what was previously assumed

Understanding these post-translational modifications provides crucial insights for drug development efforts targeting MurA and explains aspects of enzyme regulation that were previously unclear.

How Does P. amoebophila MurA Contribute to Host-Pathogen Interactions?

P. amoebophila MurA plays several important roles in host-pathogen interactions:

Peptidoglycan Recognition by Host Immune System:

• Peptidoglycan fragments are recognized by intracellular Pattern Recognition Receptors (PRRs) like Nod1 and Nod2

• These receptors detect bacterial cell wall components within host cells

• The activity of MurA contributes to the generation of these immunostimulatory molecules

Adaptation to Intracellular Environment:

• Obligate intracellular bacteria must balance peptidoglycan synthesis with immune evasion

• They are under selective pressure to reduce recognition of peptidoglycan by host PRRs

• This has led to modifications in peptidoglycan structure or its transient assembly during specific stages of the cell cycle

Inclusion Membrane Formation and Maintenance:

• P. amoebophila remains within a host-derived vesicular compartment called the inclusion

• The bacterium modifies this membrane through insertion of unique proteins (Inc proteins)

• Unlike Chlamydiaceae, P. amoebophila remains in single-cell inclusions and establishes a long-term relationship with its host

• Peptidoglycan biosynthesis likely contributes to bacterial cell integrity within these specialized compartments

Metabolic Activity in Extracellular Stages:

• P. amoebophila elementary bodies (EBs) maintain metabolic activity including D-glucose utilization

• This activity is essential for maintaining infectivity

• MurA function may be critical during these stages, particularly as EBs transition to reticulate bodies

Coordination with Cell Division:

• In bacterial systems like L. monocytogenes, MurA activity is coordinated with cell division through proteins like GpsB

• Similar coordination likely occurs in P. amoebophila during intracellular multiplication

• MurA activity must be precisely regulated to support synchronized growth of bacteria and amoebae

Understanding these interactions provides insights into the unique adaptations of P. amoebophila and related organisms to their intracellular lifestyle and suggests potential intervention points for controlling these infections.

What Techniques Can Be Used to Study MurA Structural Dynamics During Catalysis?

Several advanced techniques can be employed to investigate MurA structural dynamics during catalysis:

X-ray Crystallography:

• Captures static snapshots of MurA in different conformational states

• Previously revealed the domain closure upon substrate binding and fosfomycin interaction

• Can visualize covalent adducts formed with PEP or inhibitors

• Challenge: Requires successful crystallization of each state

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Measures the exchange of hydrogen atoms with deuterium in the protein backbone

• Regions with higher exchange rates indicate greater solvent accessibility or flexibility

• Can track conformational changes upon substrate or inhibitor binding

• Protocol: Subject MurA to D2O under various conditions (apo, substrate-bound, inhibitor-bound), quench at different time points, digest, and analyze by MS

Molecular Dynamics Simulations:

• Computationally model the dynamic behavior of MurA

• Can predict conformational changes and energy landscapes

• Useful for studying transitions between open and closed states

• Required input: High-resolution crystal structures and appropriate force fields

Fluorescence Resonance Energy Transfer (FRET):

• Monitors distance changes between fluorophores attached to specific residues

• Can track domain movements in real-time during catalysis

• Experimental approach:

  • Generate cysteine mutants at strategic positions in each domain

  • Label with fluorescent probes

  • Measure FRET efficiency changes upon substrate addition

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Provides atomic-level information on protein dynamics in solution

• Can detect subtle structural changes and identify transient interactions

• Limitations: Size constraints may require domain-specific analysis for MurA

Site-Directed Spin Labeling with Electron Paramagnetic Resonance (EPR):

• Measures distances between paramagnetic centers in a protein

• Useful for tracking large-scale conformational changes

• Approach: Introduce spin labels at specific sites and measure distance constraints

Single-Molecule Förster Resonance Energy Transfer (smFRET):

• Tracks conformational changes in individual MurA molecules

• Can reveal heterogeneity in the conformational ensemble

• Provides kinetic information on transitions between states

These techniques would be particularly valuable for understanding how P. amoebophila MurA might differ from other bacterial MurA enzymes in terms of structural dynamics and catalytic mechanism, potentially revealing unique features related to its adaptation to an intracellular lifestyle.

How Do Environmental Conditions Affect P. amoebophila MurA Activity?

Environmental conditions significantly impact P. amoebophila MurA activity through multiple mechanisms:

Temperature Effects:

• Optimal activity for most MurA enzymes occurs around 37°C

• P. amoebophila, as an amoeba symbiont, may have evolved to function at lower temperatures matching its host's environment

• Temperature fluctuations affect:

  • Enzyme kinetics (reaction rate typically increases with temperature until denaturation)

  • Protein stability and folding

  • Substrate binding affinity

pH Dependence:

Nutrient Availability:

Host Cell Interactions:

• The intracellular environment provides:

  • Osmotic protection (reducing the need for a rigid cell wall)

  • Specific ion concentrations affecting enzyme activity

  • Regulatory signals from host-pathogen communication systems

Developmental Stage-Specific Regulation:

• Expression and activity of MurA likely varies between:

  • Elementary bodies (infectious, metabolically active form)

  • Reticulate bodies (replicative form)

• This adapts cell wall synthesis to the needs of each developmental stage

Stress Responses:

• Antibiotics or host immune responses trigger stress adaptation

• These stressors may alter MurA activity through:

  • Post-translational modifications

  • Changed expression levels

  • Altered proteolytic regulation

Understanding these environmental influences is crucial for designing effective inhibitors and for interpreting experimental results when characterizing the enzyme under laboratory conditions that may differ from its native environment.

What Are the Key Differences Between MurA from P. amoebophila and Other Bacterial Species?

MurA from P. amoebophila exhibits several distinguishing features compared to MurA enzymes from other bacterial species:

Phylogenetic Context:

• P. amoebophila belongs to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) bacterial superphylum

• This places its MurA in a distinct evolutionary lineage compared to those from proteobacteria or firmicutes

• Phylogenetic analysis shows a 3 amino acid insert in RNA polymerase β subunit that is specific to Chlamydiae, Verrucomicrobia, and Lentisphaera species, suggesting similar evolutionary patterns may exist in MurA

Sequence and Structure Variations:

• Conservation of key catalytic residues (Cys115, Lys22, Asp305)

• Likely retains the two-domain architecture connected by a double-stranded linker

• May contain unique surface-exposed residues reflecting adaptation to intracellular lifestyle

• These variations could influence protein-protein interactions and regulatory mechanisms

Substrate Affinity:

• MurA from related organisms shows specific kinetic parameters:

  • Wolbachia MurA: Km = 0.03149 mM for UDP-N-acetylglucosamine and 0.009198 mM for phosphoenolpyruvate

  • P. amoebophila MurA likely has similar substrate affinity but may be optimized for its unique intracellular environment

Inhibitor Sensitivity:

• Fosfomycin sensitivity depends on conservation of the active site cysteine

• P. amoebophila MurA likely retains fosfomycin sensitivity as it maintains this conserved residue

• May exhibit different sensitivity profiles to novel inhibitor classes like pyrrolidinediones

Regulatory Interactions:

• Unlike some bacterial species, P. amoebophila has a reduced genome with potentially simplified regulatory networks

• May lack specific regulatory proteins found in other bacteria

• Could employ unique regulatory mechanisms adapted to its symbiotic lifestyle

Developmental Regulation:

• Expression patterns likely coordinated with the developmental cycle of P. amoebophila

• May be differently regulated compared to free-living bacteria or pathogenic Chlamydiaceae

• Expression during elementary body to reticulate body transition may be particularly important