Recombinant Rhodopirellula baltica Enolase (eno)

What is the function of Rhodopirellula baltica enolase in cellular metabolism?

Rhodopirellula baltica enolase (eno) catalyzes the reversible conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway. This reaction is essential for carbohydrate degradation via glycolysis in R. baltica, which serves as a model organism for aerobic carbohydrate degradation in marine systems . Proteomic analysis has identified enolase as one of the most abundantly expressed proteins in R. baltica, maintaining consistent expression across various growth conditions . This high constitutive expression, confirmed by both 2-DE gel analysis and normalized codon usage analysis, suggests enolase plays a fundamental role in R. baltica's primary metabolism . Enzyme activity assays have demonstrated that enolase maintains relatively stable activity levels (approximately 0.015-0.092 U/mg) regardless of the carbon source used for growth, indicating its central role in maintaining glycolytic flux under varying nutritional conditions .

What is the molecular structure and basic properties of R. baltica enolase?

R. baltica enolase is a 428-amino acid protein with a molecular mass of approximately 45.9 kDa, belonging to the enolase protein family . The enzyme's primary sequence has been fully characterized, and the complete amino acid sequence is available for structure-function analysis . As with other enolases, the R. baltica enzyme likely forms oligomeric structures (typically dimers) in its functional state. The enzyme requires divalent metal ions, particularly Mg²⁺, for catalytic activity, as is characteristic of the enolase superfamily . While detailed structural information specific to R. baltica enolase is still emerging, sequence analysis suggests it maintains the conserved catalytic residues and structural elements typical of bacterial enolases. The protein's relatively high molecular weight compared to some bacterial enolases may reflect specific adaptations related to R. baltica's unique cellular organization or environmental niche.

What are the kinetic parameters of recombinant R. baltica enolase and how do they compare with enolases from other organisms?

The kinetic characterization of recombinant R. baltica enolase is essential for understanding its catalytic efficiency and potential evolutionary adaptations. For comprehensive kinetic analysis, researchers should measure initial reaction velocities across a range of substrate concentrations (typically 0.1-10 mM 2-phosphoglycerate for the forward reaction) under controlled conditions. Based on enzymatic activity data, R. baltica enolase maintains activity levels between 0.015-0.092 U/mg depending on growth conditions .

When determining kinetic parameters, researchers should focus on:

• Calculating Km, Vmax, kcat, and catalytic efficiency (kcat/Km) using Michaelis-Menten kinetics

• Evaluating the effects of divalent metal ions (particularly Mg²⁺) on enzymatic activity

• Assessing pH dependence (typically pH 6.5-8.5) and temperature effects on activity

The activity measurements provided in Table 1 from research findings show relatively stable enolase activity across different carbon sources :

When comparing with other bacterial enolases, researchers should consider both phylogenetically related organisms and bacteria from similar ecological niches to identify environment-specific adaptations in the enzyme's performance .

How does recombinant R. baltica enolase activity correlate with the organism's life cycle and different cell morphologies?

R. baltica undergoes distinct morphological transitions during its life cycle, which provides a unique opportunity to study enzyme function in the context of cellular differentiation. According to microscopic examination data, R. baltica cultures show a progression of dominant cell morphologies through their growth phases :

• Early exponential phase: Dominated by swarmer and budding cells

• Transition phase: Mix of single cells, budding cells, and rosettes

• Stationary phase: Dominated by rosette formations

While enolase shows constitutive expression across different carbon sources, transcriptomic data reveals that central metabolism undergoes regulation during life cycle transitions . The transition from exponential to stationary phase involves substantial transcriptional reprogramming, with 12% of all genes showing differential expression when comparing cells at 240 hours versus 82 hours .

To investigate the relationship between enolase activity and cell morphology, researchers should implement:

• Cell cycle-specific sampling approaches, including physical separation of different cell morphologies

• Multi-omics integration combining transcriptomics, proteomics, and metabolomics

• Metabolic flux analysis using isotope-labeled glucose to trace glycolytic activity in different cell populations

The enzyme activities reported in Table 1 represent averages across mixed populations, potentially masking cell type-specific differences that could be revealed through more targeted analytical approaches .

What are the potential biotechnological applications of R. baltica enolase outside of its native glycolytic role?

R. baltica enolase has several potential biotechnological applications beyond its primary metabolic role, based on properties common to the enolase family and the unique characteristics of this marine bacterium .

• Biocatalysis in marine-mimicking conditions: R. baltica enolase likely possesses adaptations for function in marine environments, potentially including salt tolerance and activity at varying temperatures. These properties make it a candidate for biocatalytic applications requiring such conditions, particularly in the synthesis of phosphorylated compounds .

• Structural templates for protein engineering: The constitutive high-level expression of enolase in R. baltica suggests robust protein folding and stability. Understanding these properties could inform the design of stable enzymes for biotechnological applications .

• Novel substrate utilization: Investigating R. baltica enolase's substrate range might reveal capabilities beyond the canonical 2-PG/PEP interconversion. The organism's genomic context suggests potential involvement in diverse carbohydrate metabolism pathways, as R. baltica has been identified as a model organism for aerobic carbohydrate degradation in marine systems .

• Understanding moonlighting functions: Many enolases demonstrate secondary "moonlighting" functions beyond their catalytic role. R. baltica enolase might possess unique secondary functions related to the organism's unusual cell biology and environmental adaptations .

• Comparative enzymology: As a member of the deeply-branching Planctomycetes phylum, R. baltica enolase provides valuable insights into enzyme evolution and adaptation in this unique bacterial lineage .

What are the optimal conditions for expressing recombinant R. baltica enolase in E. coli?

Successful expression of functional recombinant R. baltica enolase in E. coli requires careful optimization of several parameters:

• Expression system selection:

  • For basic research: pET-based expression systems (particularly pET-28a with an N-terminal His-tag) often yield good results for bacterial enzymes

  • For higher solubility: Consider fusion tags like SUMO, MBP, or GST

  • For structural studies: Use tightly regulated expression systems to prevent inclusion body formation

• Host strain optimization:

  • BL21(DE3) and derivatives are standard choices

  • For codon optimization: Consider Rosetta or CodonPlus strains to account for codon usage differences between R. baltica and E. coli

  • For improved folding: Arctic Express or Origami strains may enhance proper folding

• Expression conditions:

  • Induction: Use lower IPTG concentrations (0.1-0.5 mM) and lower temperatures (16-25°C) for improved solubility

  • Growth media: Enriched media (like Terrific Broth) often improves yield

  • Incubation time: Extended expression (16-24 hours) at lower temperatures often yields more soluble protein

• Extraction and purification:

  • Lysis buffer optimization: Include glycerol (10-20%), stabilizing ions (Mg²⁺), and appropriate salt concentration

  • Consider including specific enolase inhibitors during purification to stabilize the protein

  • Purification via immobilized metal affinity chromatography followed by size exclusion chromatography typically yields highly pure enzyme

The search results indicate that recombinant expression of R. baltica enzymes in E. coli has been successfully achieved for several proteins from this organism, including enzymes involved in mannosylglucosylglycerate biosynthesis . Similar approaches can be adapted for enolase expression with appropriate optimization.

How can researchers accurately measure the enzymatic activity of R. baltica enolase?

Accurate measurement of R. baltica enolase activity requires careful consideration of assay conditions and potential interfering factors:

• Standard spectrophotometric assays:

  • Forward reaction (2-PG to PEP): Direct measurement of PEP formation by monitoring absorbance increase at 240 nm (ε = 1,400 M⁻¹cm⁻¹)

  • Reverse reaction (PEP to 2-PG): Coupled assay with phosphoglycerate mutase and glyceraldehyde-3-phosphate dehydrogenase, monitoring NADH oxidation at 340 nm

• Assay buffer optimization:

  • Tris-HCl or HEPES buffer (50 mM, pH 7.2-7.5)

  • Include Mg²⁺ (typically 5-10 mM MgCl₂) as enolase is a metalloenzyme

  • Standard ionic strength (typically 100 mM KCl)

  • Consider adding BSA (0.1-0.5 mg/ml) to stabilize dilute enzyme

  • Temperature: Typically 25-30°C (consider testing marine-relevant temperatures)

• Controls and validation:

  • Include enzyme-free controls to account for non-enzymatic substrate degradation

  • Commercial yeast or rabbit muscle enolase as positive controls

  • Specific enolase inhibitors (like fluoride or phosphonoacetohydroxamate) for verification

  • Substrate depletion curve analysis to confirm Michaelis-Menten kinetics

• Advanced analytical techniques:

  • NMR for direct observation of substrate/product interconversion

  • HPLC or capillary electrophoresis for simultaneous quantification of 2-PG and PEP

  • Mass spectrometry for identifying potential alternate substrates or reaction products

The data from research findings indicates that enolase activity in R. baltica cell extracts ranges from 0.015 to 0.092 U/mg depending on the growth substrate, providing a reference range for recombinant enzyme activity expectations .

What are the best approaches for studying the structure-function relationship of R. baltica enolase?

Investigating the structure-function relationship of R. baltica enolase requires a multi-faceted approach combining structural biology, biochemistry, and computational methods:

For R. baltica enolase specifically, researchers should pay particular attention to potential structural features related to the unique cellular compartmentalization in Planctomycetes and comparative analysis with enolases from other marine organisms versus terrestrial bacteria .

How does the expression pattern of enolase in R. baltica compare to other glycolytic enzymes under various growth conditions?

The expression pattern of enolase in R. baltica shows notable stability across different growth conditions, distinguishing it from some other glycolytic enzymes that demonstrate more variable expression. Based on the enzyme activity data presented in Table 1 from research findings , we can observe several important patterns:

Key observations from this comparative data include:

What distinguishes the substrate specificity of R. baltica enolase from that of other bacterial enolases?

Investigating the substrate specificity of R. baltica enolase provides insights into potential metabolic adaptations specific to this marine bacterium:

• Canonical substrate handling:

  • While the primary reaction of enolase (2-PG ⟷ PEP conversion) is highly conserved, subtle differences in kinetic parameters can reflect evolutionary adaptations

  • R. baltica enolase likely maintains the core catalytic residues found across the enolase family, including those coordinating the essential Mg²⁺ ions

  • Quantitative comparison of specificity constants (kcat/Km) for the forward and reverse reactions provides a baseline for comparison with other bacterial enolases

• Methodological approach for substrate specificity profiling:

  • Standard substrates: Measure activity with 2-PG and PEP under varying conditions (pH, temperature, salt concentration)

  • Substrate analogs: Test structural variants (e.g., 3-phosphoglycerate, phosphonopyruvate) to probe active site tolerance

  • Non-conventional substrates: Screen compounds with similar chemical features to identify potential secondary activities

  • Inhibition patterns: Compare sensitivity to known enolase inhibitors (fluoride, phosphonate analogs)

• Environmental adaptations in substrate handling:

  • Marine bacteria often face fluctuating salt concentrations that can affect enzyme-substrate interactions

  • R. baltica's unusual cell compartmentalization may result in different microenvironments for enzymatic reactions

  • The enzyme's behavior under conditions mimicking the marine environment should be compared with terrestrial bacterial enolases

• Integration with metabolic context:

  • R. baltica possesses a complete glycolytic pathway and pentose phosphate pathway

  • The relative activities of different glycolytic enzymes suggest that enolase activity is coordinated with other pathway components

  • Substrate specificity should be considered in the context of the organism's broader carbohydrate metabolism, as R. baltica is considered a model organism for aerobic carbohydrate degradation in marine systems

What are common obstacles in obtaining functional recombinant R. baltica enolase and how can they be overcome?

Researchers working with recombinant R. baltica enolase may encounter several technical challenges that can impact protein yield, solubility, and functionality. Here are the most common issues and strategies to address them:

• Codon usage bias:

  • Challenge: R. baltica, as a marine Planctomycetes, has different codon preferences than E. coli, potentially leading to translational stalling and truncated products.

  • Solution: Use codon-optimized synthetic genes or express in E. coli strains supplemented with rare tRNAs (e.g., Rosetta, CodonPlus).

• Protein solubility issues:

  • Challenge: Recombinant enolase may form inclusion bodies, particularly at high expression levels.

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.3 mM), use solubility-enhancing fusion tags (SUMO, MBP, GST), or add chemical chaperones to the growth medium.

• Metal ion requirements:

  • Challenge: Enolases require divalent metal ions (typically Mg²⁺) for proper folding and activity.

  • Solution: Supplement growth media and purification buffers with 5-10 mM MgCl₂. Avoid chelating agents such as EDTA that could strip essential metals.

• Protein stability issues:

  • Challenge: R. baltica enolase may exhibit limited stability after purification.

  • Solution: Optimize storage buffers with stabilizing agents (10-20% glycerol, 1-5 mM DTT or β-mercaptoethanol), determine optimal pH (typically 7.0-8.0), and store at -80°C in small aliquots.

• Verification of proper folding:

  • Challenge: Ensuring that recombinant enolase adopts the native conformation.

  • Solution: Combine activity assays with biophysical techniques such as circular dichroism spectroscopy, thermal shift assays, and size exclusion chromatography.

Protocols that have successfully expressed other enzymes from R. baltica, such as those described for glucosyl-3-phosphoglycerate synthase (GpgS), mannosylglucosyl-3-phosphoglycerate synthase (MggA), and MGPG phosphatase (MggB), can serve as useful starting points for optimizing expression of recombinant enolase .

How can researchers validate that recombinant R. baltica enolase maintains native-like properties?

Validating that recombinant R. baltica enolase maintains properties similar to the native enzyme is crucial for ensuring experimental relevance. Several complementary approaches can help establish the fidelity of the recombinant protein:

When validating recombinant R. baltica enolase, researchers should refer to the enzyme activity data available from cell extract studies, which provide valuable reference points for assessing whether the recombinant enzyme behaves similarly to the native form .