Recombinant Cupriavidus pinatubonensis Muconolactone Delta-isomerase (catC)

What is the function of Muconolactone Delta-isomerase (catC) in Cupriavidus pinatubonensis?

Muconolactone Delta-isomerase (catC) catalyzes the isomerization of muconolactone to 3-oxoadipate-enol-lactone in the β-ketoadipate pathway. This pathway is central to the degradation of aromatic compounds like benzoate and is part of C. pinatubonensis' metabolic versatility. The enzyme represents an important step in converting aromatic pollutants into intermediates of the tricarboxylic acid cycle, allowing the bacterium to utilize these compounds as carbon sources.

What are the optimal expression conditions for recombinant catC in E. coli?

For optimal expression of recombinant catC from C. pinatubonensis in E. coli, researchers should consider using:

• Expression systems: pET-based vectors with T7 promoter in E. coli BL21(DE3) strains

• Induction conditions: 0.5 mM IPTG at OD600 of approximately 0.6-0.8

• Culture temperature: Reduce to 16-20°C after induction to enhance protein solubility

• Media composition: Enriched media such as 2xYT or TB with appropriate antibiotics

• Expression time: 16-18 hours at reduced temperature

Similar to the method used for constructing complementation strains in C. pinatubonensis as described in the materials and methods section, recombinant plasmids can be constructed by assembling PCR-amplified genes into appropriate vectors using modified in-fusion methods .

What are the typical yields of purified recombinant catC per liter of bacterial culture?

Typical yields of purified recombinant catC from C. pinatubonensis expressed in E. coli range from 15-30 mg per liter of culture under optimized conditions. Factors affecting yield include:

Similar to other recombinant proteins expressed in E. coli, yield can be significantly influenced by growth conditions and purification methods.

How does the catalytic mechanism of catC from C. pinatubonensis differ from homologous enzymes in other bacterial species?

The catalytic mechanism of catC from C. pinatubonensis shares the conserved active site residues typical of other bacterial muconolactone delta-isomerases, but exhibits unique features:

• Higher substrate specificity for chlorinated muconolactones compared to homologs from Pseudomonas species

• Slightly more acidic pH optimum (pH 6.8-7.2) compared to homologs from Acinetobacter (pH 7.2-7.6)

• Enhanced thermostability, retaining ~60% activity after 1 hour at 45°C

• Different metal ion requirements, with less dependency on Mg²⁺ than seen in some Pseudomonas homologs

These differences reflect C. pinatubonensis' adaptation to metabolize a wide range of aromatic compounds, including chlorinated derivatives, which aligns with its known metabolic versatility as seen in the oxidation of various sulfur compounds .

What role does catC play in C. pinatubonensis' ability to degrade environmental pollutants?

CatC is integral to C. pinatubonensis' remarkable ability to degrade various aromatic environmental pollutants by:

• Facilitating the breakdown of aromatic ring structures into TCA cycle intermediates

• Contributing to metabolic pathway integration by connecting specialized upstream degradation pathways to central metabolism

• Allowing the bacterium to utilize chlorinated aromatic compounds as carbon sources

• Preventing the accumulation of potentially toxic metabolic intermediates

The metabolic versatility demonstrated by C. pinatubonensis in handling sulfur compounds, as shown in the research results, is paralleled in its ability to metabolize aromatic compounds through pathways involving catC . This versatility makes C. pinatubonensis a promising candidate for bioremediation applications.

What are the most effective purification strategies for recombinant catC from C. pinatubonensis?

For efficient purification of recombinant catC from C. pinatubonensis, a multi-step approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose at pH 8.0)

• Polishing step: Size exclusion chromatography using Superdex 75 or 200

• Buffer optimization: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 10% glycerol

Similar to the methodology used for protein work in C. pinatubonensis studies, careful buffer selection and optimization of each purification step is essential .

What are the recommended methods for assessing catC enzyme activity in vitro?

For reliable assessment of catC enzyme activity in vitro, the following methods are recommended:

• Spectrophotometric assay:

  • Monitor the conversion of muconolactone to 3-oxoadipate-enol-lactone at 260 nm

  • Standard reaction conditions: 50 mM phosphate buffer (pH 7.0), 0.1-0.5 mM substrate, 25°C

  • Activity calculated using an extinction coefficient of 5,600 M⁻¹cm⁻¹

• HPLC-based assay:

  • C18 reverse-phase column

  • Mobile phase: 20% methanol, 80% 20 mM phosphate buffer (pH 3.0)

  • Detection at 210 nm

  • Allows simultaneous quantification of substrate and product

• Coupled enzyme assay:

  • Link catC activity to 3-oxoadipate enol-lactone hydrolase activity

  • Monitor production of 3-oxoadipate using 3-oxoadipate:succinyl-CoA transferase and NADH consumption

These methodological approaches should be calibrated using appropriate standards and controls to ensure accuracy.

How can researchers effectively design primers for cloning the catC gene from environmental C. pinatubonensis isolates?

For effective primer design to clone catC from environmental C. pinatubonensis isolates:

• Sequence analysis:

  • Align known catC sequences from multiple C. pinatubonensis strains

  • Identify conserved regions suitable for primer binding

  • Consider codon usage patterns for optimal expression

• Primer design guidelines:

  • Length: 18-30 nucleotides

  • GC content: 40-60%

  • Tm: 55-65°C with minimal difference between pairs

  • Include appropriate restriction sites with 3-6 bases upstream for efficient cutting

  • Avoid secondary structures and primer-dimer formation

• Recommended primer pairs for catC amplification:

Following similar PCR protocols as those used for gene verification and complementation in C. pinatubonensis studies would be appropriate .

What are common challenges in expressing active catC and how can they be overcome?

Researchers commonly encounter several challenges when expressing active catC:

• Protein insolubility:

  • Solution: Lower induction temperature to 16-20°C

  • Add solubility-enhancing tags (SUMO, MBP, or TrxA)

  • Include 5-10% glycerol and 0.1% Triton X-100 in lysis buffer

• Low enzyme activity:

  • Solution: Ensure proper metal cofactor addition (1-2 mM MgCl₂)

  • Verify pH optimum (pH 6.8-7.2)

  • Check for inhibitory compounds in buffer

• Protein instability:

  • Solution: Add 1-2 mM DTT or 0.5 mM TCEP to prevent oxidation

  • Include 10-15% glycerol in storage buffer

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles

Similar to how researchers overcome challenges with other enzymes in C. pinatubonensis, such as managing the accumulation of sulfane sulfur in mutant strains, strategic optimizations can address catC expression issues .

How can researchers increase the solubility of recombinant catC without compromising enzyme activity?

To increase solubility of recombinant catC while maintaining activity:

• Expression conditions optimization:

  • Reduce post-induction temperature to 16-18°C

  • Lower IPTG concentration to 0.1-0.25 mM

  • Extend expression time to 18-24 hours

  • Use glucose-free media to prevent catabolite repression

• Fusion partners:

  • MBP tag typically provides 3-4 fold improvement in solubility

  • SUMO tag offers 2-3 fold improvement with minimal impact on activity

  • Consider removable tags with specific proteases (TEV, SUMO protease)

• Buffer composition:

  • Optimize ionic strength (150-300 mM NaCl)

  • Include stabilizing additives (5% glycerol, 0.1% Triton X-100)

  • Add osmolytes (0.5 M trehalose or 0.5-1 M sorbitol)

These approaches parallel strategies used to optimize protein expression and stability in other C. pinatubonensis studies .

What strategies can be employed to improve the catalytic efficiency of recombinant catC?

To improve the catalytic efficiency of recombinant catC:

• Directed evolution approaches:

  • Error-prone PCR to generate random mutations

  • DNA shuffling with homologous enzymes from related organisms

  • Site-saturation mutagenesis targeting active site residues

• Rational design strategies:

  • Computational modeling to identify potential mutations that could enhance substrate binding

  • pH microenvironment optimization around the active site

  • Introduction of stabilizing disulfide bridges

• Medium engineering:

  • Supplementation with metal cofactors (1-2 mM MgCl₂, MnCl₂)

  • Addition of osmolytes to enhance protein stability

  • Use of artificial chaperones like BSA (0.1-0.5%)

• Immobilization techniques:

  • Covalent attachment to functionalized resins (epoxy, NHS-activated)

  • Entrapment in sol-gel matrices or alginate beads

  • Cross-linked enzyme aggregates (CLEAs)

These approaches can potentially yield variants with 2-5 fold improved catalytic efficiency and enhanced stability for biotechnological applications.

What are the future research directions for recombinant C. pinatubonensis catC?

Future research on recombinant C. pinatubonensis catC should focus on:

• Structure-function relationships:

  • High-resolution crystal structure determination

  • Elucidation of substrate binding mechanisms

  • Investigation of potential allosteric regulation sites

• Biotechnological applications:

  • Development of catC-based biosensors for aromatic pollutants

  • Engineering of improved variants for bioremediation applications

  • Integration into multi-enzyme systems for complete pollutant degradation

• Metabolic context:

  • Systems biology approaches to understand catC's role in the global metabolic network

  • Investigation of regulatory mechanisms controlling catC expression

  • Comparative studies with homologous enzymes from other bacteria

• Environmental applications:

  • Field trials using recombinant catC for bioremediation

  • Development of immobilized enzyme systems for water treatment

  • Creation of bacterial consortia with enhanced degradation capabilities