Recombinant Escherichia coli O9:H4 S-adenosylmethionine decarboxylase proenzyme (speD)
Description
Enzymatic Function and Biological Role
SpeD catalyzes the decarboxylation of S-adenosylmethionine (AdoMet) to produce dcAdoMet, a prerequisite for spermidine biosynthesis via spermidine synthase (SpeE) . This reaction is essential for bacterial growth and stress adaptation. Key features include:
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Autocatalytic processing: The proenzyme self-cleaves at an internal serine residue to generate a pyruvoyl cofactor, forming α- and β-subunits .
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Substrate specificity: Canonical SpeD exclusively processes AdoMet, but neofunctionalized homologs in other bacteria (e.g., Candidatus Marinimicrobia) exhibit L-arginine or L-ornithine decarboxylase activity .
Recombinant Expression in E. coli
While E. coli O9:H4 SpeD-specific studies are sparse, recombinant SpeD workflows in E. coli systems (e.g., BL21 strains) provide a template:
Expression and Purification
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Vector systems: Polycistronic plasmids (e.g., pST44) enable co-expression of multi-subunit complexes, though SpeD is typically expressed as a single gene .
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Tagging: N-terminal His-tags facilitate purification via affinity chromatography, yielding >90% purity .
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Yield: Recombinant SpeD constitutes ~52% of total bacterial protein under optimal induction (1 mM IPTG, 37°C, 5 hr) .
| Parameter | Value | Source |
|---|---|---|
| Molecular weight | ~38 kDa (proenzyme) | |
| Optimal pH | 8.0 (Tris/PBS buffer) | |
| Storage stability | -80°C with 50% glycerol | |
| Activity (kₐₜ/Kₘ) | 770 M⁻¹s⁻¹ (L-arginine analogs) |
Biochemical Characterization
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Kinetic analysis: Coupled assays measuring CO₂ release confirm activity. For example, Ca. Marinimicrobia SpeD exhibits a kₐₜ/Kₘ of 770 ± 37 M⁻¹s⁻¹ for L-arginine .
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LC-MS validation: Tribenzoylated spermidine derivatives are detected post-reaction, confirming functional reconstitution in speD-deficient E. coli .
Applications and Pathogenic Context
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Metabolic engineering: SpeD supports polyamine production in industrial E. coli strains .
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Virulence linkage: While O9:H4 is primarily commensal, hybrid pathotypes (e.g., STEC/EAEC) may exploit polyamines for biofilm formation or immune evasion .
Challenges and Future Directions
Product Specs
Target Background
KEGG: ecx:EcHS_A0124
