Recombinant Acinetobacter sp. 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (gpmI), partial

What is the difference between 2,3-bisphosphoglycerate-dependent and independent phosphoglycerate mutases?

Phosphoglycerate mutases (PGMs) catalyze the interconversion of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis pathways, but they differ in their catalytic mechanisms:

• 2,3-bisphosphoglycerate-dependent PGM (GpmA): Utilizes 2,3-bisphosphoglycerate as a catalytic cofactor. These enzymes operate through a phosphoenzyme (Ping Pong) mechanism where a phosphate group is transferred from 2,3-bisphosphoglycerate to the enzyme and then to the substrate .

• 2,3-bisphosphoglycerate-independent PGM (GpmI): Does not require 2,3-bisphosphoglycerate but has an absolute and specific requirement for Mn²⁺ ions. These enzymes are typically larger (approximately 57 kDa compared to ~27 kDa for GpmA) and show high pH sensitivity .

The distinction is important for experimental design as metal chelation during purification may affect GpmI activity but not GpmA activity.

What methods are recommended for expression and purification of recombinant Acinetobacter sp. gpmI?

For successful expression and purification of recombinant Acinetobacter sp. gpmI, the following protocol is recommended:

• Cloning: Amplify the gpmI gene using PCR with gene-specific primers incorporating appropriate restriction sites .

• Vector selection: Clone the amplified gene into an expression vector containing a promoter system (such as T7 or LacZ) and a suitable affinity tag (most commonly His-tag) .

• Expression host: Transform the recombinant plasmid into a compatible E. coli strain, such as BL21(DE3) for efficient protein expression .

• Induction conditions: Grow transformed cells to mid-log phase (OD₆₀₀ of 0.6-0.8) before inducing protein expression with IPTG (typically 0.5-1.0 mM) at 25-30°C to minimize inclusion body formation .

• Purification: Harvest cells by centrifugation, lyse by sonication, and purify using nickel-affinity chromatography for His-tagged proteins .

• Quality control: Verify protein purity using SDS-PAGE analysis and confirm identity by proteomic analysis such as mass spectrometry .

Crucial consideration: Maintain Mn²⁺ in buffers during purification as GpmI has an absolute requirement for this metal cofactor .

How is phosphoglycerate mutase activity measured in laboratory settings?

Phosphoglycerate mutase activity can be measured using several methods:

Direct spectrophotometric assay:

• Monitor the conversion of 3-phosphoglycerate to 2-phosphoglycerate by coupling with enolase, pyruvate kinase, and lactate dehydrogenase.

• Measure the decrease in NADH absorbance at 340 nm, which corresponds to enzymatic activity .

Radioisotope-based assays:

• Use ³²P-labeled or ¹⁴C-labeled substrates to track phosphate transfer or carbon flow through the reaction .

• These assays can distinguish between different PGM mechanisms by comparing the rates of ³²P and ¹⁴C exchange at chemical equilibrium .

Kinetic analysis protocol:

• Prepare reaction buffer containing the substrate (typically 3-phosphoglycerate) at various concentrations (10-500 μM)

• For GpmI, include MnCl₂ (typically 1-5 mM) as a cofactor

• Initiate reaction by adding purified enzyme and monitor product formation

• Calculate kinetic parameters (Km, Vmax, kcat) using appropriate software

For Acinetobacter sp. gpmI, typical kinetic values include high affinity for substrate (Km in the range of 90-150 μM) with kcat/Km values of approximately 5.30 × 10⁴ M⁻¹s⁻¹ .

How does metal dependency affect the function and stability of phosphoglycerate mutase in Acinetobacter species?

Metal dependency significantly impacts both the function and structural stability of phosphoglycerate mutase in Acinetobacter species:

Functional effects:

• Mn²⁺ is an absolute requirement for catalytic activity of 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (GpmI) .

• Metal chelation completely abolishes enzymatic activity, which cannot be restored by other divalent cations .

• The metal ion coordinates substrate binding and facilitates phospho-transfer during catalysis.

Structural stability:

• Circular dichroism studies have shown that while the binding of Mn²⁺ causes minimal changes in secondary structure, it significantly increases thermal stability of the enzyme .

• Thermal unfolding analysis revealed that Mn²⁺ binding caused a large increase in enzyme stability, while substrate binding (3-phosphoglycerate) did not significantly affect stability .

Experimental evidence:
When exposed to metal-chelating agents (such as EDTA), GpmI activity decreases dramatically, whereas GpmA activity remains largely unaffected. This differential response provides a useful experimental approach to distinguish between the two enzyme types in biochemical assays .

In Staphylococcus aureus, which possesses both GpmI and GpmA, metal limitation by calprotectin (CP) induces a 40-fold increase in gpmA expression while gpmI expression remains unchanged, suggesting evolutionary adaptations to metal-limited environments during infection .

What structural features distinguish 2,3-bisphosphoglycerate-independent phosphoglycerate mutase and what methods are used to determine its crystal structure?

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (GpmI) has distinctive structural features that can be elucidated through specific crystallographic methods:

Structural features:

• Molecular weight of approximately 57 kDa (compared to ~27 kDa for GpmA)

• Monomeric quaternary structure (unlike some GpmA enzymes which form dimers)

• Absence of signature sequences characteristic of Types A, B, and C nonspecific bacterial acid phosphatases

• Contains a metal-binding site specifically coordinating Mn²⁺

Crystallization methods:

• Crystal growth conditions: Successful crystallization has been achieved using the oil-microbatch method at 291K with ammonium sulfate as precipitating agent .

• Diffraction quality: Crystals typically diffract X-rays to beyond 2.5 Å resolution .

• Space group determination: Crystals of Bacillus stearothermophilus GpmI belong to the orthorhombic space group C2221 with unit cell dimensions: a = 58.42, b = 206.08, c = 124.87 Å, and α = β = γ = 90.0° .

• Structure determination approaches:

  • Multiwavelength anomalous dispersion (MAD) using selenomethionyl-substituted protein

  • Molecular replacement when suitable homologous structures are available

Experimental protocol for selenomethionyl protein production:

• Express recombinant protein in a methionine auxotrophic E. coli strain (such as B834)

• Grow in minimal medium supplemented with selenomethionine

• Purify using standard affinity chromatography

• Crystallize under conditions similar to native protein but with reducing agents to prevent selenomethionine oxidation

For Pyrococcus horikoshii OT3 phosphoglycerate mutase, crystals were obtained in space group R32 with unit cell parameters a = 155.62, c = 230.35 Å, diffracting to 2.2 Å resolution, suggesting a dimeric arrangement in the asymmetric unit (VM value of 3.0 ų Da⁻¹) .

How does phosphoglycerate mutase contribute to bacterial virulence and survival under stress conditions?

Phosphoglycerate mutase plays critical roles in bacterial pathogenesis and stress adaptation through multiple mechanisms:

Virulence mechanisms:

• Metabolic adaptation: In Staphylococcus aureus, the metal-independent phosphoglycerate mutase (GpmA) enables glycolytic flux to continue during host-imposed metal starvation, supporting bacterial survival .

• Energy production: By maintaining glycolysis under stress conditions, phosphoglycerate mutase ensures ATP generation for virulence factor expression and bacterial replication .

• Biofilm formation: In Acidovorax citrulli, the BdpmAc (2,3-bisphosphoglycerate-dependent phosphoglycerate mutase) mutant exhibited decreased biofilm formation, suggesting this enzyme's role in surface attachment and persistence .

Stress adaptation:

Experimental evidence of virulence contribution:
In mouse infection models with wild-type S. aureus versus ΔgpmA mutants, mice infected with ΔgpmA lost significantly less weight and showed reduced bacterial burden, demonstrating the enzyme's importance in pathogenesis .

Comparative proteomic analysis of wild-type Acidovorax citrulli versus bdpmAc knockout mutant revealed differential abundance of proteins involved in:

• Motility

• Cell wall/membrane/envelope biogenesis

• Carbohydrate metabolism

These findings suggest phosphoglycerate mutase has pleiotropic effects extending beyond its canonical metabolic role, making it a potential target for anti-virulence therapeutic development .

What kinetic mechanisms have been elucidated for phosphoglycerate mutase and how are they experimentally determined?

The kinetic mechanisms of phosphoglycerate mutases have been extensively studied using specialized experimental approaches:

Possible mechanisms:

• Phosphoenzyme (Ping Pong) mechanism: A phosphate group is transferred from 2,3-diphosphoglycerate to the enzyme, forming a phosphorylated enzyme intermediate, which then transfers the phosphate to the substrate .

• Sequential mechanism: An intermolecular transfer of phosphate from 2,3-diphosphoglycerate directly to the substrates .

• Intramolecular transfer: Direct transfer of phosphate between positions within a single substrate molecule .

Experimental methods for mechanism determination:

• Induced-transport tests:

  • Using ¹⁴C-labeled substrates to detect potential co-transport phenomena

  • With ³²P-labeled substrates to monitor phosphate transfer patterns

• Isotope exchange rate comparison:

  • Comparing exchange rates of ³²P and ¹⁴C-labeled substrates at chemical equilibrium

  • For 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle, ¹⁴C-labeled substrates exchange at twice the rate of ³²P-labeled substrates, indicating a phosphoenzyme mechanism

• Steady-state kinetic analysis:

  • Double-reciprocal plots to distinguish between sequential and ping-pong mechanisms

  • Product inhibition patterns to validate the proposed mechanism

Experimental findings:
Studies with the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle provided conclusive evidence for a phosphoenzyme mechanism with a remarkably rapid isomerization rate constant of at least 4×10⁶s⁻¹ . The intramolecular transfer of phosphate and intermolecular transfer between substrate molecules were completely excluded based on the experimental data .

The phosphoenzyme mechanism suggests evolutionary relationships between phosphoglycerate mutases and other phosphatases, particularly 2,3-diphosphoglycerate phosphatases .

What genomic and comparative proteomic approaches can be used to characterize phosphoglycerate mutase function in Acinetobacter species?

Comprehensive characterization of phosphoglycerate mutase function in Acinetobacter species requires integrated genomic and proteomic approaches:

Genomic approaches:

• Genome sequencing and annotation:

  • Whole genome sequencing to identify phosphoglycerate mutase genes

  • Comparative genomics to understand gene organization and regulation

  • Analysis of GC content (typically around 42.8% for Acinetobacter species)

• Phylogenetic analysis:

  • 16S rRNA gene sequencing for species identification

  • GBDP (Genome BLAST Distance Phylogeny) analysis to establish evolutionary relationships

  • Type strain genome server (TYGS) analysis to identify novel species

• Gene knockout strategies:

  • EZ-Tn™ insertional mutagenesis for functional characterization

  • PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes for species identification

Proteomic approaches:

• Comparative proteomic analysis workflow:

  • Culture wild-type and mutant strains under identical conditions

  • Extract total proteins and perform tryptic digestion

  • Analyze using LC-MS/MS

  • Identify differentially abundant proteins

  • Perform functional classification and pathway analysis

• Protein characterization methods:

  • SDS-PAGE for molecular weight determination

  • Western blotting for expression verification

  • Mass spectrometry for protein identification

Integration of multi-omics data:
Analysis of Acidovorax citrulli BdpmAc mutant using comparative proteomics revealed differential abundance of proteins involved in:

• Motility

• Cell wall/membrane biogenesis

• Carbohydrate metabolism

• Stress response

These findings were correlated with phenotypic assays demonstrating that the mutant:

• Exhibited decreased virulence

• Could not grow with fructose or pyruvate as a sole carbon source

• Showed reduced biofilm formation and twitching motility

• Displayed increased osmotic tolerance

The combined genomic and proteomic approach provides comprehensive insights into the pleiotropic functions of phosphoglycerate mutase beyond its canonical metabolic role.