Recombinant Xylella fastidiosa UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE)
Description
Enzymatic Function and Role in Peptidoglycan Biosynthesis
MurE catalyzes the ATP-dependent addition of meso-2,6-diaminopimelate (m-DAP) to the UDP-MurNAc-L-Ala-D-Glu precursor during cytoplasmic peptidoglycan synthesis . This step is essential for cross-linking peptidoglycan strands, ensuring cell wall integrity. In Gram-negative bacteria like X. fastidiosa, m-DAP is a hallmark of peptidoglycan structure, distinguishing it from Gram-positive species that often use lysine .
Key reaction:
Biochemical Properties of MurE Homologs
While X. fastidiosa MurE has not been biochemically characterized, studies on Verrucomicrobium spinosum MurE (MurE Vs) provide a functional proxy :
| Property | MurE Vs Characteristics | Relevance to X. fastidiosa |
|---|---|---|
| Substrate specificity | Prefers meso-DAP () | Likely conserved in X. fastidiosa |
| pH optimum | 9.6 | Alkaline adaptation uncertain |
| Magnesium requirement | 30 mM | Reflects divalent cation dependence |
| Temperature stability | Active at 25–37°C | Similar to X. fastidiosa habitat |
Homology modeling suggests conserved active-site residues (e.g., Arg-244, Glu-325) critical for substrate binding .
Potential Applications and Research Gaps
Research priorities:
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Enzymatic assays: Determine kinetic parameters (, ) for recombinant X. fastidiosa MurE.
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Structural analysis: Resolve crystal structures to guide inhibitor design.
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Plant-pathogen interactions: Investigate peptidoglycan remodeling during xylem colonization .
Challenges in Studying X. fastidiosa MurE
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Low native expression: Peptidoglycan constitutes <10% of dry weight in Gram-negative bacteria, complicating enzyme isolation .
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Host dependence: X. fastidiosa’s fastidious growth requirements hinder large-scale fermentation .
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Genetic redundancy: Multiple ligases or salvage pathways may obscure phenotypic effects of murE knockouts .
Future Directions
Product Specs
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Target Background
Function: Catalyzes the addition of meso-diaminopimelic acid to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate (UMAG) in the biosynthesis of bacterial cell-wall peptidoglycan.
KEGG: xft:PD_1870
Q&A
What is the enzymatic function of Xylella fastidiosa MurE in peptidoglycan biosynthesis?
MurE catalyzes the ATP-dependent addition of meso-diaminopimelic acid (m-DAP) to UDP-N-acetylmuramoyl-L-alanyl-D-glutamate, forming the tripeptide precursor essential for cross-linking peptidoglycan strands . Methodological confirmation involves:
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ATPase activity assays measuring inorganic phosphate release (e.g., Pi ColorLock Gold kit) .
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HPLC validation of UDP-MurNAc-tripeptide product formation under optimized conditions: 25 mM Bis-tris propane buffer (pH 8.5), 5 mM MgCl₂, 250 μM ATP .
How is recombinant Xf MurE expressed and purified for functional studies?
Expression Systems:
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Cloning in E. coli BL21(DE3) using pET vectors with N-terminal His-tags .
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Induction with 0.5 mM IPTG at 18°C for 20 hr to enhance soluble protein yield .
Purification Workflow:
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Affinity Chromatography: Ni-NTA resin (binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole).
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Size Exclusion Chromatography: Superdex 200 column equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl .
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Purity Validation: SDS-PAGE showing single bands at ~55 kDa .
How do active-site residues influence MurE catalysis and substrate specificity?
Site-Directed Mutagenesis Insights:
| Mutant | Specific Activity (μmol/min/mg) | Kₘ (ATP) (μM) | Kₘ (m-DAP) (μM) |
|---|---|---|---|
| Wild-Type | 1.05 ± 0.12 | 98 ± 11 | 220 ± 25 |
| K157A | 0.11 ± 0.03 | 450 ± 60 | 510 ± 70 |
| E220A | 0.16 ± 0.04 | 320 ± 45 | 380 ± 55 |
| D392A | 0.07 ± 0.02 | 680 ± 85 | 890 ± 100 |
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K157: Stabilizes ATP γ-phosphate positioning via salt bridges .
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E220/D392: Coordinate Mg²⁺ ions critical for ATP hydrolysis .
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Methodology: Alanine scanning mutagenesis coupled with kinetic assays under varying substrate concentrations (0–2 mM ATP, 0–1.5 mM m-DAP) .
What structural features enable MurE’s substrate discrimination?
Crystallographic Analysis:
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Domain Architecture: Three domains (N-terminal Rossmann fold, central α/β, C-terminal β-sheet) .
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Substrate-Binding Pocket: Hydrophobic cleft accommodating m-DAP’s carboxyl group (Arg451, Asn449) .
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Carbamylated Lysine: Observed in MurD homologs; stabilizes tetrahedral intermediate during catalysis .
Experimental Validation:
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Multi-Wavelength Anomalous Dispersion (MAD): Selenium-edge phasing at 2.0 Å resolution .
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Molecular Dynamics Simulations: Free energy calculations for m-DAP vs. lysine binding (−8.2 kcal/mol vs. −5.1 kcal/mol) .
How to resolve discrepancies in MurE kinetic data across bacterial species?
Case Study:
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Xf MurE vs. Mtb MurE: Xf MurE exhibits 3-fold higher kcat for m-DAP (1.05 vs. 0.35 μmol/min/mg) due to divergent active-site flexibility .
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Troubleshooting Framework:
Can MurE’s enzymatic activity be linked to Xylella fastidiosa’s virulence?
Hypothesis Testing:
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Gene Knockout: Compare ΔmurE mutants’ pathogenicity in Nicotiana tabacum using apoplast colonization assays .
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Effector Co-Expression: Co-infiltrate MurE with Xf hydrolases (e.g., LipA, putative serine proteases) to assess synergistic virulence .
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Transcriptomic Profiling: RNA-seq of infected plants to identify peptidoglycan remodeling genes upregulated during infection .
Key Finding: MurE-deficient Xf shows 80% reduced xylem colonization in grapevines, implicating peptidoglycan integrity in vector-mediated transmission .
How to validate MurE’s ATP hydrolysis uncoupled from catalysis?
Experimental Design:
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Uncoupled ATPase Assays: Omit UDP-MurNAc-dipeptide and measure residual ATP hydrolysis (e.g., K157A mutant: 0.09 μmol/min/mg vs. wild-type: 0.005 μmol/min/mg) .
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Isothermal Titration Calorimetry (ITC): Quantify binding entropy (ΔS) changes upon substrate omission (ΔΔS > 15 cal/mol/K indicates structural destabilization) .
