Recombinant Escherichia coli O7:K1 Agmatinase (speB)

Introduction to Recombinant Escherichia coli Agmatinase (SpeB)

Agmatinase (EC 3.5.3.11), encoded by the speB gene, is a member of the ureohydrolase superfamily. In E. coli, it regulates cellular polyamine levels by converting agmatine—a decarboxylated derivative of arginine—into putrescine, a precursor for spermidine and spermine . Recombinant SpeB is produced via heterologous expression systems for structural and functional studies, enabling detailed mechanistic insights .

Active Site Architecture

• Metal Binding: Two manganese ions (Mn²⁺) coordinate a hydroxide ion for nucleophilic attack on agmatine .

• Critical Residues:

  • Asp153 and His163: Stabilize the transition state and participate in proton transfer .

  • Trp68: Stabilizes the substrate’s amino group via cation-π interactions .

• Dynamic Loops: Loops A (residues 158–162) and B (residues 188–191) gate substrate entry and specificity .

Crystallographic Data

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Mechanism

1. Substrate Binding: Agmatine coordinates Mn²⁺ via its guanidinium group.

2. Hydrolysis: A metal-activated hydroxide ion attacks the guanidinium carbon, yielding urea and putrescine .

Kinetic Parameters

Mutagenesis studies reveal the impact of active-site residues on catalysis:

Source:

Loop B mutants (e.g., F188T/E191D) reduce $k_{cat}$ by 100-fold, underscoring its role in transition-state stabilization .

Functional Roles in Bacterial Physiology

• Polyamine Biosynthesis: Putrescine supports biofilm formation, acid resistance, and stress adaptation .

• Host-Microbe Interactions:

  • SpeB deletion in E. coli increases intracellular agmatine, altering host lipid metabolism and extending lifespan in C. elegans via dietary restriction-like pathways .

  • Transcriptomic studies show speB induction under anaerobic conditions and rumen fluid exposure in E. coli O157:H7, linking it to virulence and environmental adaptation .

Biotechnological Relevance

• Enzyme Engineering: Loop modifications (e.g., G158T/C159S) shift substrate specificity toward arginine, suggesting potential for industrial polyamine production .

• Drug Target: Agmatinase inhibitors could modulate polyamine levels in pathogens or cancer cells .

Key Discoveries

• Structural Plasticity: Molecular dynamics simulations reveal conformational flexibility in substrate-binding loops, critical for catalysis .

• Metabolic Cross-Talk: SpeB activity intersects with arginine deiminase pathways, influencing nitrogen metabolism .

References

1. Chitrakar et al. (2021). PLoS One 16: e0248991. DOI: 10.1371/journal.pone.0248991

2. Transcriptome analysis of E. coli O157:H7 under host-like conditions. PDF

3. Maturana et al. (2021). Int J Mol Sci 22: 4769. DOI: 10.3390/ijms22094769

4. Kinetics and structural analysis of EcAGM mutants. MDPI

5. Pryor et al. (2019). PMC6736778. Host-microbe-drug-nutrient interactions.

6. UniProtKB entry for SpeB (Q1R791). UniProt