Recombinant Pseudomonas putida UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE)

Basic Research Questions

• What is the function of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE) in Pseudomonas putida?

  MurE is a cytoplasmic enzyme that catalyzes a critical step in bacterial cell wall peptidoglycan biosynthesis. Specifically, it adds meso-diaminopimelic acid to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate. This reaction is essential for the formation of the peptide cross-links in the peptidoglycan layer, which provides structural integrity to the bacterial cell wall. In P. putida, as in other Gram-negative bacteria, this enzyme is crucial for cell survival, making it an interesting target for basic research and potential antimicrobial development .

• What expression systems are recommended for producing recombinant P. putida murE?

  For recombinant expression of P. putida murE, yeast expression systems have shown good results based on available research. When designing expression experiments, consider the following methodology:

  • Vector selection: Use vectors with strong, inducible promoters compatible with your expression host

  • Expression host: While yeast has been documented , E. coli expression systems can also be viable alternatives

  • Purification strategy: Include an affinity tag (typically His-tag) for efficient purification

  • Buffer optimization: Use buffers containing divalent cations (Mg²⁺ or Mn²⁺) which are required for enzyme activity

  The expression protocol should include cell lysis optimization, as proper release of the recombinant protein is crucial for downstream applications. Protein yield can vary between 5-20 mg/L of culture depending on optimization parameters.

• How is the activity of recombinant P. putida murE typically assayed?

  The activity of recombinant P. putida murE can be assayed using several methods:

  1. ATP consumption assay: Measures the hydrolysis of ATP during the ligase reaction

  2. HPLC-based assay: Detects the formation of UDP-MurNAc-tripeptide

  3. Coupled enzyme assay: Links ADP production to NADH oxidation for spectrophotometric detection

  A standard activity assay reaction mixture contains:

  Component	Concentration
  Purified murE	0.1-1 μM
  UDP-MurNAc-L-Ala-D-Glu	0.1-0.5 mM
  meso-DAP	0.1-1 mM
  ATP	2-5 mM
  MgCl₂	10-15 mM
  Buffer (typically Tris-HCl, pH 8.0)	50-100 mM

  Note that higher concentrations of ATP and UDP-sugar substrates can be inhibitory for Mur synthetase activities, suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway .

Advanced Research Questions

• What genetic engineering approaches are effective for modifying the murE gene in P. putida?

  Several advanced genetic engineering approaches have proven effective for modifying the murE gene in P. putida:

  1. ReScribe system: This combines multiplex recombineering with CRISPR-Cas9 counterselection, allowing efficient gene editing. The system has shown editing efficiencies of 90-100% in P. putida strains, even with recalcitrant targets .

  2. Recombinase-based methods: Rec2 and PapRecT recombinases can be used for ssDNA recombineering, especially when combined with transient inhibition of the native mismatch repair system by co-expressing a dominant-negative allele of mutL .

  Implementation protocol:

  • Design oligonucleotides targeting the murE gene (60-90 nucleotides, without phosphorothioate bonds which don't increase efficiency in P. putida)

  • Express recombinase (Rec2 or PapRecT) under thermoinducible promoter

  • Transform cells with oligonucleotides after heat induction

  • Apply CRISPR-Cas9 counterselection to eliminate non-edited cells

  For multiplex editing, efficiency levels reach 95.2% after three recombineering cycles when targeting three genes simultaneously .

• How does the kinetic behavior of P. putida murE differ from murE in other bacterial species?

  P. putida murE exhibits distinctive kinetic properties compared to homologous enzymes from other bacteria:

  Parameter	P. putida murE	E. coli murE	M. tuberculosis murE
  K<sub>m</sub> for UDP-MurNAc-L-Ala-D-Glu	0.15-0.3 mM	0.1-0.2 mM	0.5-0.8 mM
  K<sub>m</sub> for meso-DAP	0.02-0.05 mM	0.01-0.03 mM	0.1-0.3 mM
  K<sub>m</sub> for ATP	0.3-0.5 mM	0.2-0.4 mM	0.5-0.8 mM
  Optimal pH	7.5-8.0	7.6-8.2	8.0-8.5
  Divalent cation requirement	Absolute	Absolute	Absolute

  All Mur synthetases require divalent cations for activity, but P. putida murE shows distinctive inhibition patterns at higher substrate concentrations. The negative feedback at elevated concentrations of ATP and UDP-sugar substrates suggests a stringent regulatory mechanism in the peptidoglycan biosynthetic pathway that may be species-specific .

  Research has also revealed that P. putida murE, like other Mur synthetases, may be regulated by serine/threonine protein kinases (PknA and PknB), adding another layer of complexity to its kinetic regulation .

• How can structural analysis of P. putida murE contribute to antimicrobial drug discovery?

  Structural analysis of P. putida murE provides several advantages for antimicrobial drug discovery:

  1. Identification of catalytic residues: Crystallographic studies of homologous enzymes have revealed key catalytic residues, including a carbamylated lysine residue in the active site . This knowledge can be applied to structure-based drug design targeting P. putida murE.

  2. Substrate binding pocket analysis: Understanding the three-domain structure of murE and how it binds UDP-MurNAc-L-Ala-D-Glu and meso-DAP can guide the development of competitive inhibitors.

  3. Species-specific features: Structural differences between murE from different bacterial species can be exploited to develop narrow-spectrum antibiotics.

  Research methodology should include:

  • Protein crystallization in complex with substrates, products, or inhibitors

  • X-ray diffraction analysis (aim for resolution <2.0 Å)

  • Molecular dynamics simulations to identify flexible regions

  • Virtual screening of compound libraries against the active site

  A significant finding from homologous structures is that murE contains a structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor , which could be targeted for selective inhibition.

• What role does murE play in the EDEMP cycle in P. putida, and how does this differ from other metabolic pathways?

  While murE's primary function is in peptidoglycan biosynthesis, understanding its relationship to P. putida's central metabolism, particularly the EDEMP cycle, provides insight into metabolic interconnections:

  P. putida utilizes an unusual glycolytic cycle called the EDEMP cycle, which merges components of the Entner-Doudoroff and pentose phosphate pathways along with gluconeogenic reactions from upper glycolysis . This cycle is significantly different from the standard Embden-Meyerhof-Parnas pathway found in many bacteria.

  The connection between murE and central metabolism involves:

  1. UDP-glucose derivatives: The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate substrate originates from UDP-glucose, connecting cell wall synthesis to carbohydrate metabolism

  2. Amino acid utilization: The incorporation of D-glutamate and meso-DAP links to amino acid metabolism

  3. Energy consumption: ATP requirements create connections to the cell's energy status

  P. putida's distinctive central metabolism, including its ability to grow on diverse carbon sources and its unique EDEMP cycle, may influence the regulation and activity of murE compared to other bacteria. This understanding can be important when developing metabolic engineering strategies that might affect cell wall biosynthesis .

• How can one optimize multiplex genome editing of P. putida to target murE alongside other peptidoglycan biosynthesis genes?

  Optimizing multiplex genome editing to target murE alongside other peptidoglycan biosynthesis genes requires a systematic approach:

  1. ReScribe system implementation: The ReScribe system combines Rec2-mediated recombineering with CRISPR-Cas9 counterselection and has shown efficiencies of 95.2% when targeting three genes simultaneously after three cycles .

  Optimization protocol:

  Step	Method	Duration	Efficiency
  Single gene editing	Standard recombineering	6 days/mutation	8.3% average
  Single gene with ReScribe	Single-targeting ReScribe	3 days/mutation	90.5% average
  Multiplex editing	Multiplex ReScribe	3 days for multiple mutations	77.8% average

  1. Target selection considerations:

     • Select targets in the division/cell wall (dcw) operon, which contains murE and other peptidoglycan synthesis genes

     • Consider co-transcription patterns when targeting multiple genes

     • Avoid targeting genes with overlapping functions to prevent lethality

  2. Oligonucleotide design optimization:

     • Use 60-90 nucleotide oligos without phosphorothioate bonds

     • Target the lagging strand of DNA replication

     • Introduce silent mutations in PAM sequences to prevent repeated CRISPR cleavage

  The rate of off-target mutations was found to be approximately 1.17 per recombineering cycle , which should be considered when planning multiplex editing experiments.

• What are the experimental challenges in studying potential post-translational modifications of P. putida murE?

  Studying post-translational modifications (PTMs) of P. putida murE presents several experimental challenges:

  1. Phosphorylation analysis: Research suggests that Mur synthetases interact with Ser/Thr protein kinases (PknA and PknB) . Detecting and characterizing phosphorylation sites requires:

     • Phosphoproteomic approaches (TiO₂ enrichment followed by LC-MS/MS)

     • Site-directed mutagenesis of potential phosphorylation sites

     • In vitro kinase assays with purified kinases

  2. Other potential PTMs:

     • Active site carbamylation: Evidence from homologous proteins suggests lysine carbamylation in the active site

     • Detecting this modification requires careful mass spectrometry under native conditions

  3. Methodological challenges:

     • Maintaining PTMs during protein extraction and purification

     • Distinguishing between in vivo modifications and artifacts

     • Correlating PTMs with functional effects on enzyme activity

  4. Functional analysis of PTMs:

     • Creating phosphomimetic mutations (Ser/Thr to Asp/Glu)

     • Measuring kinetic parameters of modified versus unmodified enzyme

     • Determining the effect of PTMs on protein-protein interactions

  Recent work on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation provides methodological frameworks that can be adapted for P. putida murE studies .

• How can interspecies comparative analysis of murE contribute to understanding peptidoglycan evolution in pseudomonads?

  Comparative analysis of murE across different Pseudomonas species and other bacteria provides insights into peptidoglycan evolution:

  1. Sequence and structural analysis:

     • Multiple sequence alignment of murE from different pseudomonads (P. putida, P. aeruginosa, P. fluorescens)

     • Identification of conserved domains and species-specific features

     • Phylogenetic tree construction to trace evolutionary relationships

  2. Functional conservation assessment:

     • Cross-species complementation experiments

     • Comparing substrate specificity and kinetic parameters

     • Analyzing differences in regulation (transcriptional, post-translational)

  3. Ecological adaptations:

     • Comparing murE functionality in pseudomonads from different ecological niches

     • Analyzing co-evolution with cell wall architecture

  A particularly interesting finding is that while P. putida and P. aeruginosa share many metabolic features, their central metabolism differs significantly. P. putida operates an unusual EDEMP cycle for glucose metabolism, which may influence how cell wall synthesis integrates with central metabolism across different pseudomonads .

  Understanding these differences can provide insights into how peptidoglycan biosynthesis has adapted to various ecological niches and evolutionary pressures, potentially revealing novel targets for species-specific antimicrobial development.