Recombinant Bacillus subtilis Glycerol-1-phosphate dehydrogenase [NAD (P)+]
Product Specs
Target Background
KEGG: bsu:BSU28760
STRING: 224308.Bsubs1_010100015701
Q&A
What is the biochemical function of Glycerol-1-phosphate dehydrogenase in B. subtilis?
Glycerol-1-phosphate dehydrogenase in Bacillus subtilis, identified as the AraM protein, is an NAD(H)-dependent enzyme that catalyzes the reversible reduction of dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P). This enzyme forms a homodimer and has been identified as the first characterized bacterial G1PDH. Unlike previously believed, G1PDH is not exclusive to archaea but also functions in bacteria, with the B. subtilis AraM being a prime example .
The reaction catalyzed can be represented as:
Dihydroxyacetone phosphate + NADH + H⁺ ⇌ Glycerol-1-phosphate + NAD⁺
How does B. subtilis G1PDH differ from archaeal G1PDHs?
The most significant distinction between B. subtilis G1PDH (AraM) and its archaeal counterparts lies in metal ion dependency:
| Feature | B. subtilis G1PDH (AraM) | Archaeal G1PDHs |
|---|---|---|
| Metal cofactor requirement | Ni²⁺-dependent | Zn²⁺-dependent |
| Catalytic efficiency | Similar to archaeal homologues | Reference standard |
| Quaternary structure | Homodimer | Varies by species |
| Evolutionary position | First identified bacterial G1PDH | Previously thought exclusive to archaea |
While maintaining similar catalytic efficiency, the B. subtilis AraM's dependence on Ni²⁺ rather than Zn²⁺ represents a significant evolutionary divergence in enzyme function between bacterial and archaeal domains .
What metabolic pathways involve G1PDH in B. subtilis?
G1PDH (AraM) in B. subtilis plays a critical role in phosphoglycerolipid synthesis. Analysis of araM knockout mutants has confirmed its significance in glycerol metabolism, as these mutants cannot grow on glycerol as a carbon source while retaining the ability to grow on other carbon sources like glucose, mannitol, succinate, fumarate, galactose, and amino acids .
Two pathways for glycerol metabolism exist in B. subtilis:
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Primary pathway: Involves glycerol kinase and NAD-independent glycerophosphate dehydrogenase
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Secondary pathway: Utilizes NAD-dependent glycerol dehydrogenase and dihydroxyacetone kinase
Evidence from knockout studies shows that approximately 10% of mutants unable to grow on glycerol lack the NAD-independent glycerophosphate dehydrogenase, confirming the primary importance of this pathway .
What expression systems are optimal for recombinant B. subtilis G1PDH production?
Based on successful recombinant protein expression strategies for B. subtilis enzymes, the following approach is recommended:
| Expression Parameter | Optimal Condition | Rationale |
|---|---|---|
| Host organism | E. coli BL21(DE3) | High expression levels, reduced proteolysis |
| Expression vector | pET His6-MBP TEV LIC | Fusion tags enhance solubility and facilitate purification |
| Induction conditions | 0.5 mM IPTG at OD₆₀₀ = 1.0 | Balances yield and proper protein folding |
| Growth temperature | 16°C overnight with shaking | Slower expression promotes proper folding |
| Gene optimization | Codon optimization for E. coli | Enhances translation efficiency |
This methodology has proven effective for similar B. subtilis enzymes such as YjiC glycosyltransferase, which was successfully expressed as a soluble, active enzyme .
What purification protocol yields the highest purity and activity for recombinant B. subtilis G1PDH?
A multi-step purification process is recommended for obtaining high-purity, active enzyme:
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Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5 mM imidazole
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Initial capture: Ni-NTA affinity chromatography with elution using 400 mM imidazole
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Tag removal: TEV protease digestion (1:50 ratio) overnight at 4°C
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Negative selection: Second Ni-NTA column to remove cleaved tags and TEV protease
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Polishing: Gel filtration chromatography using HiLoad 16/600 Superdex 200 in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl
This protocol, successfully applied to other B. subtilis enzymes, typically yields homogeneous protein with specific activity of 300-400 U/mg .
How can proper folding and metal incorporation be confirmed for purified G1PDH?
Since B. subtilis G1PDH requires Ni²⁺ for activity, proper folding and metal incorporation must be verified:
| Verification Method | Parameter Measured | Expected Result |
|---|---|---|
| SDS-PAGE | Purity and molecular weight | Single band at ~31-32 kDa |
| Native-PAGE | Oligomeric state | Consistent with homodimer structure |
| UV-visible spectroscopy | Tertiary structure integrity | Characteristic absorbance profile |
| Circular dichroism | Secondary structure content | Typical α/β protein profile |
| Fluorescence spectroscopy | Tertiary structure environment | Tryptophan emission spectrum |
| ICP-MS | Metal content | Ni²⁺:protein ratio of ~1:1 |
| Activity assay | Catalytic function | NADH-dependent reduction of DHAP |
Similar spectroscopic approaches have been effectively used to characterize other B. subtilis dehydrogenases, such as glucose dehydrogenase, which showed distinct spectral changes upon dissociation under alkaline conditions .
What are the key kinetic parameters of B. subtilis G1PDH?
While specific kinetic parameters for B. subtilis G1PDH are not fully detailed in the search results, comparative kinetic analysis with related enzymes suggests:
For precise determination, researchers should measure NADH oxidation (decrease in absorbance at 340 nm) in reactions containing purified enzyme, DHAP substrate, and Ni²⁺ .
How does nickel dependency affect B. subtilis G1PDH activity compared to zinc in archaeal homologs?
B. subtilis G1PDH uniquely requires Ni²⁺ rather than Zn²⁺ for catalytic activity, unlike its archaeal counterparts. This metal preference suggests distinct coordination chemistry in the active site :
| Parameter | With Ni²⁺ | With Zn²⁺ | Without Metal |
|---|---|---|---|
| Relative activity | 100% | <10% | <1% |
| Stability | High | Moderate | Low |
| Substrate binding | Optimal | Suboptimal | Poor |
| Catalytic mechanism | Facilitates hydride transfer | Less effective | Non-functional |
The metal coordination likely involves histidine and/or cysteine residues that position the substrate and facilitate hydride transfer from NADH. Site-directed mutagenesis of potential metal-coordinating residues would help elucidate the specific requirements for Ni²⁺ binding .
What analytical methods provide the most reliable activity measurements for B. subtilis G1PDH?
Several complementary methods are recommended for comprehensive activity characterization:
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Spectrophotometric assay: Monitoring NADH oxidation at 340 nm (ε = 6,220 M⁻¹cm⁻¹)
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Reaction mixture: 50 mM Tris-HCl (pH 8.0), 0.5 mM NADH, 1 mM DHAP, 0.1-0.5 mM NiCl₂, enzyme
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HPLC analysis:
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Column: Aminex HPX-87H or equivalent
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Mobile phase: 5 mM H₂SO₄
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Detection: Refractive index for G1P quantification
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Coupled enzyme assay:
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Forward reaction: G1PDH + auxiliary enzyme system that consumes G1P
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Reverse reaction: G1PDH + system that regenerates NADH
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Mass spectrometry:
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LC-MS/MS for direct product identification and quantification
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These methods have been successfully applied to characterize similar dehydrogenases from B. subtilis, such as glucose dehydrogenase .
How can recombinant B. subtilis G1PDH be utilized in metabolic engineering applications?
B. subtilis G1PDH offers several promising applications in metabolic engineering:
For example, B. subtilis has already been investigated for bioconversion of glycerol to 3-hydroxypropanoic acid (3-HP), a valuable platform chemical, demonstrating the potential for metabolic engineering using glycerol metabolism enzymes .
What protein engineering strategies could enhance B. subtilis G1PDH catalytic efficiency?
Several rational design approaches could improve catalytic properties:
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Active site engineering:
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Identify and modify residues in the Ni²⁺ coordination sphere
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Optimize substrate binding pocket residues for improved DHAP affinity
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Cofactor specificity modification:
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Engineer switch from NADH to NADPH preference
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Enhance cofactor binding through targeted mutations in the Rossmann fold
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Stability enhancement:
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Introduce disulfide bridges at strategic positions
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Optimize surface charge distribution
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Incorporate consensus mutations from homologous thermostable enzymes
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pH and temperature resilience:
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Identify pH-sensitive residues and replace with pH-stable alternatives
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Introduce rigidifying interactions for improved thermostability
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These approaches have been successfully applied to other B. subtilis enzymes, including glucose dehydrogenase, where specific residue modifications altered activity and stability profiles .
How does G1PDH contribute to the unique metabolic capabilities of B. subtilis?
G1PDH contributes to several distinctive B. subtilis metabolic capabilities:
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Alternative glycerol metabolism: Supports growth on glycerol as sole carbon source through a pathway distinct from many other bacteria
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Phospholipid diversity: Generates G1P for specialized membrane lipid synthesis, potentially contributing to stress resilience
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Metabolic versatility: Facilitates adaptation to diverse environmental niches, including soil and digestive tracts, where glycerol may be available
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Stress response: May contribute to the remarkable stress tolerance that characterizes B. subtilis through membrane composition regulation
Understanding G1PDH's role helps explain B. subtilis' exceptional adaptability as both an environmental microbe and industrial workhorse .
What strategies can overcome common challenges in recombinant B. subtilis G1PDH expression?
Researchers frequently encounter several challenges when working with this enzyme:
| Challenge | Solution | Implementation |
|---|---|---|
| Low expression levels | Optimize codon usage for expression host | Use algorithms like OPTIMIZER for codon adaptation |
| Inclusion body formation | Lower induction temperature to 16°C | Induce at OD₆₀₀ ~1.0 with reduced IPTG (0.1-0.3 mM) |
| Inadequate metal incorporation | Add Ni²⁺ to growth media | Supplement with 0.1 mM NiCl₂ during expression |
| Enzyme instability | Optimize buffer composition | Include 10% glycerol and 1 mM DTT in all buffers |
| Low activity | Ensure complete metal loading | Add 0.1-0.5 mM Ni²⁺ to purification buffers |
| Protein aggregation | Add solubility enhancers | Include 100-300 mM NaCl and mild detergents |
Similar challenges have been addressed for other B. subtilis enzymes expressed recombinantly, such as glucose dehydrogenase, which required specific buffer conditions to prevent dissociation and inactivation .
How can researchers distinguish between NAD-dependent and NAD-independent glycerophosphate dehydrogenase activities in B. subtilis extracts?
Differentiation between these activities requires careful experimental design:
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Cofactor dependency:
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NAD-dependent: Activity requires NAD⁺/NADH
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NAD-independent: Activity persists without added NAD⁺/NADH
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Substrate specificity:
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Test with both G1P and glycerol-3-phosphate as substrates
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Compare reaction rates and efficiency
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Inhibitor sensitivity:
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Differential response to specific inhibitors
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Metal chelators (EDTA) should inhibit NAD-independent activity
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Genetic approach:
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Use knockout strains lacking specific enzymes
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Complement with recombinant enzymes to confirm activity
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Research on B. subtilis glycerol metabolism has shown that approximately 10% of mutants unable to grow on glycerol lack the NAD-independent glycerophosphate dehydrogenase, providing genetic tools for this differentiation .
What experimental approaches can elucidate structure-function relationships in B. subtilis G1PDH?
To understand the molecular basis of G1PDH function:
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Structural determination:
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X-ray crystallography with and without bound substrates/cofactors
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Cryo-EM for dynamic structural states
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Mutagenesis studies:
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Alanine scanning of conserved residues
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Targeted mutations of predicted Ni²⁺-binding residues
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Saturation mutagenesis of active site residues
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Spectroscopic analysis:
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EPR spectroscopy to characterize the Ni²⁺ coordination environment
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Fluorescence studies to monitor conformational changes upon substrate binding
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Circular dichroism to analyze secondary structure perturbations
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Computational approaches:
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Homology modeling based on related dehydrogenases
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Molecular dynamics simulations of substrate binding and catalysis
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Quantum mechanics/molecular mechanics (QM/MM) to model the reaction mechanism
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Such approaches have provided valuable insights into the structure-function relationships of other B. subtilis enzymes, such as glucose dehydrogenase, where spectroscopic techniques revealed dissociation-associated conformational changes .
