Recombinant Bacillus weihenstephanensis S-adenosylmethionine decarboxylase proenzyme (speH)
Description
Table 1: Key Biochemical Properties
| Property | Details |
|---|---|
| Gene ID (NCBI) | 22938365 |
| Uniprot ID | A9VGY7 (mature enzyme), A9VJP4 (proenzyme) |
| Protein Size | 142 amino acids (mature chain), 65 residues (proenzyme fragment) |
| Catalytic Activity | AdoMet → decarboxylated AdoMet (EC 4.1.1.50) |
Expression Systems and Recombinant Production
The enzyme is commercially available in multiple expression platforms:
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Yeast: Full-length protein (CSB-YP001658BON) with >85% purity .
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E. coli: Biotinylated variants (CSB-EP001658BON-B) for advanced assays .
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Baculovirus/Mammalian: For eukaryotic post-translational modifications .
Table 2: Recombinant Variants and Applications
| Expression System | Product Code | Applications |
|---|---|---|
| Yeast | CSB-YP001658BON | WB, ELISA, enzymatic assays |
| E. coli | CSB-EP001658BON | Structural studies, kinetics |
| Baculovirus | CSB-BP001658BON | High-yield production |
Functional and Mechanistic Insights
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Autoprocessing Mechanism: Serine residue at position 68 undergoes nucleophilic attack to form an ester intermediate, followed by cleavage to generate the active pyruvoyl group . Mutations (e.g., S68A) abolish activity .
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Thermostability: Retains functionality across a broad temperature range, consistent with B. weihenstephanensis’s psychrotolerant nature .
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Substrate Specificity: Unlike some homologs, speH does not exhibit neofunctionalized activity (e.g., L-ornithine/arginine decarboxylation) .
Research Applications
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Polyamine Biosynthesis Studies: Used to investigate spermidine/spermine regulation in extremophiles .
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Drug Development: Serves as a model for designing inhibitors targeting pyruvoyl-dependent decarboxylases in pathogens .
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Industrial Biotechnology: Optimized for high-yield spermidine production in bioreactors .
Product Specs
Target Background
KEGG: bwe:BcerKBAB4_4412
STRING: 315730.BcerKBAB4_4412
Q&A
What are the critical parameters for successful heterologous expression of recombinant speH in E. coli?
The expression of speH requires careful optimization of induction temperature and codon usage patterns. While the native B. weihenstephanensis speH gene (UniProt A9VJP4) contains GC-rich regions (62% GC content in residues 1–65) , successful expression in E. coli BL21(DE3) demands:
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Induction temperature: 18–20°C to prevent inclusion body formation
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Vector design: pET-28a(+) modified with Bacillus-optimized ribosome binding sites
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Supplementation: 2 mM MgCl₂ and 0.5 mM pyridoxal phosphate in autoinduction media
Empirical data from baculovirus systems show 85% solubility when using C-terminal His-tag configurations , though N-terminal tags may interfere with proenzyme processing.
How does the proenzyme structure influence speH decarboxylase activation?
The speH proenzyme undergoes autocatalytic cleavage between Gly65 and Ser66 to generate functional α/β subunits . Structural analysis reveals three activation prerequisites:
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pH threshold: <6.8 for proper conformation of the catalytic pocket
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Ionic strength: 150 mM NaCl stabilizes subunit interaction
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Redox environment: 5 mM DTT maintains Cys42 in reduced state
Comparative studies with B. cereus speH show 40% slower activation kinetics in the psychrotolerant variant, correlating with a distorted β-hairpin motif (residues 58–64) .
What validation methods confirm speH purity and structural integrity post-purification?
A tiered analytical approach is recommended:
Discrepancies between theoretical (31.5 kDa) and observed (28 kDa) molecular weights arise from atypical migration patterns of proenzyme isoforms .
How to resolve batch-to-batch variability in speH specific activity during scale-up?
Systematic troubleshooting should address:
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Proteolytic degradation: Add 1 mM PMSF during cell lysis if aberrant 22 kDa bands appear on SDS-PAGE
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Incomplete proenzyme processing: Extend autoactivation to 48 hr at 4°C with 0.1 mM S-adenosylmethionine
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Metal ion contamination: Chelate with 5 mM EDTA before ion-exchange chromatography
Case study data show that implementing a post-purification refolding step (20 mM Tris-HCl, pH 7.0 + 0.5 M arginine) increases active yield by 73% .
What experimental strategies differentiate speH allosteric regulation artifacts from true kinetic cooperativity?
Employ a three-pronged approach:
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Substrate titration (0.1–10 mM S-adenosylmethionine) with Hill coefficient analysis
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Site-saturation mutagenesis of putative allosteric site (Asp129Glu/Asn variants)
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Molecular dynamics simulations comparing open/closed states (AMBER 20 force field)
Recent simulations reveal a novel gating mechanism in the psychrotolerant speH where loop residues 89–94 act as a thermal sensor, explaining apparent cooperativity below 15°C .
What kinetic assay design minimizes artifacts in speH activity measurements?
Implement a coupled assay system:
Reaction Scheme
S-adenosylmethionine → decarboxylated product + CO₂
CO₂ + PEP → oxaloacetate (PEP carboxylase)
Oxaloacetate + NADH → malate + NAD⁺ (malate dehydrogenase)
Optimized Conditions
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50 mM MES buffer, pH 6.2
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0.2 U/mL PEP carboxylase (Sigma P9278)
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5 mM MgCl₂
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ΔA₃₄₀ monitored at 10°C
This method achieves 98% correlation with direct HPLC quantification while enabling real-time kinetics .
How to engineer speH for enhanced thermostability without compromising cold-activity?
Apply consensus sequence analysis across psychrotolerant Bacillus orthologs:
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Identify conserved residues in (mesophilic) - (psychrotolerant) multiple sequence alignment
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RosettaDDG calculations for ΔΔG stability predictions
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Site-directed mutagenesis targeting flexible regions (RMSF >1.5 Å in MD simulations)
Notable success with triple mutant T54S/Q128R/K201E increased T₅₀ from 42°C to 49°C while maintaining 80% activity at 7°C .
