Recombinant Chlorate reductase subunit beta

What is chlorate reductase and what role does the beta subunit play?

Chlorate reductase (Clr) is a heterotrimeric enzyme consisting of α, β, and γ subunits (also referred to as ClrA, ClrB, and ClrC) that catalyzes the reduction of chlorate to chlorite in chlorate-respiring bacteria (CRB). The beta subunit (ClrB) contains four sequence motifs that could bind Fe-S clusters and plays a crucial role in electron transfer within the enzyme complex . Unlike the α and γ subunits, the ClrB protein does not contain a signal peptide for export to the periplasm, suggesting it is translocated as a folded protein together with ClrA via the Tat system using a "hitchhiker" mechanism .

Which organisms naturally produce chlorate reductase?

Chlorate reductase has been identified and characterized in several chlorate-respiring bacteria, most notably:

• Ideonella dechloratans

• Pseudomonas chloritidismutans

• Pseudomonas sp. strain PK

• Dechloromonas species

These organisms can couple growth to the reduction of chlorate as the sole electron acceptor . Unlike perchlorate-reducing bacteria (PRB) that can reduce both perchlorate and chlorate, chlorate-reducing bacteria (CRB) can only reduce chlorate to chlorite .

How is the clrB gene organized within the chlorate reductase gene cluster?

The chlorate reductase genes in I. dechloratans are organized in an operon structure designated as clrABDC:

• clrA encodes the α-subunit containing a molybdopterin cofactor

• clrB encodes the β-subunit with Fe-S clusters

• clrD encodes a specific chaperone likely involved in molybdopterin cofactor insertion

• clrC encodes the γ-subunit, which is a cytochrome b

Downstream of these genes, two additional open reading frames (ORFs) have been identified: one encoding a soluble cytochrome c (cyc), which may be involved in electron transport, and another encoding a protein involved in molybdopterin cofactor maturation (mobB) .

How is chlorate reductase expression regulated?

Chlorate reductase expression is oxygen-dependent. Both enzyme activity assays and mRNA analyses by real-time quantitative reverse transcription (qRT-PCR) have shown that chlorate reductase is expressed during aerobic growth, but transfer to anaerobic conditions with chlorate results in significantly enhanced enzyme activities and mRNA levels .

Notably, chlorate reductase showed approximately 200 times higher enzyme activity in anaerobically induced cells, whereas the increase in mRNA was only about 10-fold, suggesting additional mechanisms influence enzyme activity beyond transcriptional regulation . The absence of oxygen is necessary for this induction, as chlorate addition under aerobic conditions produces neither increased enzyme activities nor higher relative levels of mRNA .

Which expression systems are suitable for recombinant production of chlorate reductase beta subunit?

Based on studies with other subunits of chlorate reductase, E. coli expression systems using vectors such as pET-3a or pGEX-2T have been employed successfully . For the beta subunit specifically, considerations should include:

• Codon optimization for E. coli if the source organism has high GC content (as seen with I. dechloratans)

• Expression as a fusion protein (e.g., GST-fusion) to improve solubility and facilitate purification

• Co-expression with molecular chaperones that may aid in proper folding of the Fe-S cluster-containing protein

• Expression under anaerobic conditions to enhance proper insertion of Fe-S clusters

The presence of Fe-S clusters in ClrB makes it particularly challenging for recombinant expression, as proper assembly of these clusters often requires specific cellular machinery.

What challenges are associated with heterologous expression of ClrB?

Several challenges have been reported when expressing components of chlorate reductase:

• Formation of inclusion bodies (as observed with ClrC subunit expressed as a GST fusion)

• Proper insertion of Fe-S clusters (for ClrB)

• High GC content of the source genes (like in I. dechloratans) creating difficulties in PCR amplification and requiring buffers optimized for GC-rich templates

• Potential proteolytic degradation of the expressed protein

• Limited solubility of the recombinant protein

Similar issues were encountered with the C subunit of I. dechloratans chlorate reductase, which was expressed as a GST fusion protein but formed inclusion bodies. This required purification under denaturing conditions, followed by refolding and reconstitution with heme .

What purification strategies are effective for recombinant ClrB?

Based on approaches used for other chlorate reductase subunits and similar Fe-S proteins, the following purification strategies may be effective:

• For soluble recombinant ClrB:

  • Affinity chromatography (if expressed with tags)

  • Ion exchange chromatography

  • Size exclusion chromatography

• For inclusion body-derived ClrB:

  • Solubilization using denaturing agents (urea or guanidinium chloride)

  • Purification under denaturing conditions using ion exchange chromatography

  • Controlled refolding by gradual removal of the denaturant

  • Reconstitution of Fe-S clusters

For the C subunit of chlorate reductase, inclusion bodies were washed in low ionic strength buffer, solubilized in urea, and further purified by cation exchange chromatography, yielding approximately 50 mg protein per liter of cell culture .

How can you verify proper folding and Fe-S cluster incorporation in recombinant ClrB?

Several analytical techniques can be employed to verify proper folding and Fe-S cluster incorporation:

• UV-visible spectroscopy: Fe-S clusters exhibit characteristic absorption spectra

• Electron paramagnetic resonance (EPR) spectroscopy: Provides information about the oxidation state and environment of Fe-S clusters

• Circular dichroism (CD) spectroscopy: Assesses secondary structure elements

• Iron and sulfur content analysis: Quantification of Fe and S atoms per protein molecule

• Activity assays: Functional tests to confirm that the recombinant protein is active, often as part of a reconstituted enzyme complex

For the C subunit of I. dechloratans chlorate reductase, UV-vis spectroscopy was used to characterize the refolded and heme-reconstituted fusion protein, showing spectral characteristics similar to those of native chlorate reductase .

What methods can be used to assess the activity of recombinant ClrB?

Since ClrB functions as part of the heterotrimeric chlorate reductase complex, activity assays typically require:

• Reconstitution of the complete enzyme complex: Combining recombinant ClrA, ClrB, and ClrC subunits

• Chlorate reduction assay: Measuring chlorate consumption or chlorite production

• Electron transfer assays: Assessing the ability of ClrB to accept/donate electrons using artificial electron donors/acceptors

• Fe-S cluster redox potential measurements: Using cyclic voltammetry or other electrochemical methods

Typically, chlorate reductase activity can be measured spectrophotometrically by monitoring the oxidation of reduced methyl viologen (artificial electron donor) at 578 nm in the presence of chlorate .

How does the activity of recombinant ClrB compare to native enzyme?

When comparing recombinant to native enzymes, several parameters should be assessed:

• Kinetic parameters: Km values for chlorate and other substrates. Native chlorate reductase has Km values of approximately 27 μM for perchlorate and <5 μM for chlorate

• Substrate specificity: Besides chlorate, the native enzyme can reduce nitrate, iodate, and bromate at considerable rates

• pH and temperature optima: Determining if these match the native enzyme

• Stability: Assessing thermal and storage stability

For meaningful comparisons, the recombinant ClrB should be incorporated into a functional enzyme complex with the other subunits, as the individual subunit may not show activity on its own.

What is known about the structure of ClrB and how it relates to function?

While detailed structural information specifically for chlorate reductase beta subunit is limited, inferences can be made based on homologous proteins:

• ClrB contains four sequence motifs predicted to bind Fe-S clusters

• It belongs to the type II subgroup of the dimethyl sulfoxide (DMSO) reductase family

• It shows similarity to subunits in selenate reductase (Ser), dimethyl sulfide dehydrogenase (Ddh), and ethylbenzene dehydrogenase (Edh)

The Fe-S clusters in ClrB likely mediate electron transfer between the electron donor system and the molybdopterin cofactor in the catalytic A subunit. Understanding the exact arrangement and properties of these clusters would provide insights into the electron transfer mechanism within the enzyme.

How can site-directed mutagenesis be used to study ClrB function?

Site-directed mutagenesis can be a powerful approach to study ClrB function:

• Target selection: Based on sequence alignment with homologous proteins, identify conserved residues potentially involved in:

  • Fe-S cluster coordination

  • Subunit interaction

  • Protein folding and stability

• Mutation strategies:

  • Conservative substitutions (e.g., cysteine to serine) to assess the role of specific residues in Fe-S cluster binding

  • Charge reversal or removal to study electrostatic interactions

  • Deletion or insertion mutations to investigate structural elements

• Functional assessment:

  • Express and purify mutant proteins

  • Characterize Fe-S cluster content

  • Measure electron transfer capabilities

  • Assess ability to form functional complexes with other subunits

  • Determine impact on enzymatic activity

This approach has been used successfully with similar Fe-S proteins to map electron transfer pathways and identify critical functional residues.

How does ClrB participate in the electron transport pathway?

The electron transport in chlorate respiration involves:

• Electrons from the membrane-bound respiratory complexes are transferred to soluble electron carriers in the periplasm

• These electrons are then used by chlorate reductase (including ClrB) to reduce chlorate to chlorite

• Finally, chlorite dismutase catalyzes the decomposition of chlorite to chloride and oxygen

In I. dechloratans, a soluble periplasmic cytochrome c (cyt c-Id1) has been identified as an electron donor to chlorate reductase . ClrB, with its Fe-S clusters, likely plays a crucial role in accepting electrons from this cytochrome and transferring them to the molybdenum cofactor in the catalytic A subunit.

What experimental approaches can determine protein-protein interactions in the electron transport pathway?

Several techniques can be employed to study the interaction between ClrB and other components:

• Co-immunoprecipitation: Using antibodies against ClrB to pull down interacting proteins

• Surface plasmon resonance: Measuring real-time binding kinetics

• Isothermal titration calorimetry: Determining thermodynamic parameters of binding

• Cross-linking coupled with mass spectrometry: Identifying residues at the interaction interface

• Yeast two-hybrid or bacterial two-hybrid assays: Screening for potential protein partners

• Electron transfer kinetics: Measuring rates of electron transfer between purified components

For example, studies with the universal immunoprobe for (per)chlorate-reducing bacteria showed that antibodies raised against chlorite dismutase could detect chlorate-reducing bacteria across different phylogenetic groups, indicating common epitopes and potential protein interactions .

How can protein engineering be applied to modify ClrB properties?

Protein engineering approaches for ClrB could target:

• Improved stability: Introducing stabilizing mutations or disulfide bridges

• Enhanced activity: Modifying the microenvironment of Fe-S clusters or improving electron transfer pathways

• Altered substrate specificity: Engineering the interface between ClrB and ClrA to change the substrate preference

• Improved recombinant expression: Removing problematic sequences that lead to misfolding or aggregation

Directed evolution approaches, such as error-prone PCR and DNA shuffling, could also be employed to generate ClrB variants with desired properties. The resulting variants would need to be screened for activity in a reconstituted enzyme system.

What are the potential applications of recombinant ClrB in bioremediation research?

Recombinant ClrB, as part of a functional chlorate reductase complex, could have several applications in bioremediation research:

• Perchlorate/chlorate contamination: Development of enzymatic systems for degradation of (per)chlorate pollutants

• Biosensors: Creation of electrochemical biosensors for detection of chlorate in environmental samples

• Biocatalysis: Use in selective oxidation/reduction reactions of industrial relevance

• Model systems: Study of microbial adaptation to anthropogenic compounds

The ability of chlorate reductase to reduce other oxyanions such as nitrate, iodate, and bromate also suggests potential applications in the bioremediation of these contaminants.

How does ClrB compare to similar subunits in related enzymes?

Comparative analysis reveals several similarities and differences between ClrB and related proteins:

This comparative approach can provide insights into the evolution of these enzymes and their adaptation to different substrates.

What can be learned from heterologous expression of ClrB in different host organisms?

Heterologous expression in different hosts can reveal:

• Host-specific factors affecting expression: Identifying limiting factors such as rare codons, chaperones, or Fe-S cluster assembly machinery

• Post-translational modifications: Determining if specific modifications occur or are required

• Functional conservation: Testing if the recombinant protein can complement mutants lacking similar proteins

• Folding requirements: Identifying conditions that promote proper folding in different cellular environments

For example, the C subunit of I. dechloratans chlorate reductase was expressed as a GST fusion protein in E. coli, but required refolding and reconstitution with heme to produce a protein with spectral characteristics similar to the native enzyme .

How can low expression levels of recombinant ClrB be addressed?

Several strategies can improve expression levels:

• Codon optimization: Adapting the coding sequence to the codon preference of the host organism

• Promoter selection: Testing different promoters to find optimal expression levels

• Induction conditions: Optimizing temperature, inducer concentration, and induction time

• Media composition: Adding supplements that promote Fe-S cluster formation (iron, cysteine)

• Expression strain selection: Using strains optimized for expressing Fe-S proteins

• Growth under anaerobic or microaerobic conditions: Limiting oxidative stress during expression

When expressing the C subunit of chlorate reductase, adding the heme precursor δ-aminolevulinic acid and FeSO4 to the culture increased enzyme activity . Similar approaches might be beneficial for ClrB expression.

What methods can resolve protein aggregation and inclusion body formation?

If recombinant ClrB forms inclusion bodies, several approaches can be considered:

• Preventing aggregation:

  • Lower expression temperature (e.g., 16-20°C)

  • Lower inducer concentration

  • Co-expression with molecular chaperones

  • Fusion with solubility-enhancing tags (MBP, SUMO, TrxA)

  • Addition of stabilizing agents to growth medium

• Recovering protein from inclusion bodies:

  • Washing inclusion bodies with low ionic strength buffer

  • Solubilization using chaotropic agents (urea, guanidinium chloride)

  • Purification under denaturing conditions

  • Controlled refolding through gradual removal of denaturant

  • Reconstitution with Fe-S clusters

This approach was successful for the C subunit of chlorate reductase, which was expressed as a GST fusion protein, purified from inclusion bodies, refolded, and reconstituted with heme .