Recombinant Synechocystis sp. Putative carboxymethylenebutenolidase (sll1298)

What is the predicted function of the putative carboxymethylenebutenolidase (sll1298) in Synechocystis sp. PCC 6803?

The putative carboxymethylenebutenolidase (sll1298) in Synechocystis sp. PCC 6803 likely functions as a hydrolase enzyme, similar to its human homolog CMBL. Based on comparative analysis with human CMBL, it may be involved in hydrolyzing ester bonds, particularly in structures containing carboxymethylenebutenolidase-like domains. Human CMBL is known to function as a bioactivating enzyme that hydrolyzes prodrugs such as olmesartan medoxomil, converting them to pharmacologically active metabolites . In cyanobacteria, this enzyme may play roles in metabolic pathways involving similar chemical transformations, potentially participating in degradation pathways of complex organic compounds or secondary metabolites.

For researchers interested in functional characterization, a methodological approach would involve:

• Sequence alignment with characterized carboxymethylenebutenolidases

• Structure prediction to identify catalytic residues

• Heterologous expression and purification

• Substrate screening assays with potential ester-containing substrates

• Site-directed mutagenesis of predicted catalytic residues (e.g., cysteine residues similar to Cys132 in human CMBL)

What expression systems are most effective for producing recombinant sll1298 protein?

For effective recombinant expression of sll1298 from Synechocystis, researchers should consider both heterologous expression in E. coli and homologous expression in Synechocystis itself.

E. coli Expression System:

• Use BL21(DE3) strain for high-level expression, similar to the approach used for human CMBL

• Express with an N-terminal His₆-tag for purification

• Optimize codon usage for E. coli if yield is low

• Consider expression at lower temperatures (16-20°C) to improve protein folding

• Purify using nickel affinity chromatography

Synechocystis Expression System:

• For homologous expression, consider both chromosome integration and plasmid-based approaches

• For chromosomal integration, design homology regions flanking the integration site of interest

• For plasmid-based expression, use a self-replicating vector with an appropriate promoter

• Consider using the rhamnose-inducible Prha promoter, which allows precise induction of gene expression

Expression Comparison Table:

When optimizing expression, monitor protein production through Western blotting and enzymatic activity assays. For detection purposes, consider adding epitope tags (FLAG, His) to facilitate protein visualization and purification .

How can I determine if my recombinant sll1298 protein is correctly folded and active?

To assess proper folding and activity of recombinant sll1298:

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure elements

  • Thermal shift assays to evaluate protein stability

  • Size exclusion chromatography to confirm monomeric/oligomeric state

  • Limited proteolysis to assess compact folding

• Activity Assays:

  • Based on human CMBL function, design hydrolysis assays using ester-containing substrates

  • Test activity against para-nitrophenyl acetate or similar colorimetric substrates

  • Assess activity with potential physiological substrates in Synechocystis

  • Include controls with heat-inactivated enzyme and known inhibitors

• Functional Validation:

  • Create site-directed mutants of predicted catalytic residues (e.g., cysteine residues similar to Cys132 in human CMBL)

  • Compare activity between wild-type and mutant proteins

  • A dramatic reduction in activity upon mutation of key residues would confirm their catalytic importance

• Determining Optimal Conditions:

  • Test activity across pH range (6.0-9.0)

  • Evaluate temperature optima (20-40°C)

  • Assess buffer and salt requirements

  • Screen for cofactor dependencies

The activity of human CMBL is significantly reduced when the Cys132 residue is mutated to alanine or serine , suggesting that similar cysteine residues might be critical for sll1298 activity. Identifying and mutating the equivalent residue in sll1298 would provide valuable insights into its catalytic mechanism.

What are the optimal conditions for purifying recombinant sll1298 while maintaining enzymatic activity?

The purification of recombinant sll1298 requires careful consideration of conditions to preserve enzymatic activity:

Purification Protocol:

• Cell Lysis:

  • Use gentle lysis methods (e.g., lysozyme treatment followed by mild sonication)

  • Include protease inhibitors (PMSF, EDTA, or commercial cocktails)

  • Maintain low temperature (4°C) throughout processing

  • Buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol

• Affinity Chromatography:

  • For His-tagged constructs, use nickel affinity gel as described for human CMBL

  • Apply gradual imidazole gradient (20-250 mM) for elution

  • Consider tag removal with thrombin if the tag affects activity

  • Collect fractions and assess protein purity via SDS-PAGE

• Secondary Purification:

  • Ion exchange chromatography (IEX) based on theoretical pI

  • Size exclusion chromatography for final polishing and buffer exchange

  • Concentrate protein using 10 kDa MWCO centrifugal filters

• Activity Preservation:

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to protect thiol groups

  • Add 10-20% glycerol to prevent freeze-thaw damage

  • Avoid multiple freeze-thaw cycles

  • Store in small aliquots at -80°C for long-term storage

• Quality Control:

  • Measure specific activity before and after each purification step

  • Determine protein concentration using Bradford assay

  • Verify purity by SDS-PAGE (>95% homogeneity)

  • Confirm identity by mass spectrometry

Stability Assessment Table:

The purification strategy should be validated by comparing activity measurements throughout the process to ensure minimal loss of function during purification.

How can I determine the substrate specificity of sll1298 in comparison to human CMBL?

Determining substrate specificity requires a systematic approach comparing sll1298 with human CMBL:

• Substrate Panel Screening:

  • Test model substrates: p-nitrophenyl esters with varying acyl chain lengths

  • Evaluate medically relevant prodrugs known to be hydrolyzed by human CMBL (olmesartan medoxomil, faropenem medoxomil, lenampicillin)

  • Screen potential physiological substrates from Synechocystis metabolic pathways

  • Include structurally diverse lactones and ester-containing compounds

• Kinetic Parameter Determination:

  • Measure Km, kcat, and catalytic efficiency (kcat/Km) for each substrate

  • Plot Michaelis-Menten curves to visualize enzyme-substrate relationships

  • Compare kinetic parameters between sll1298 and human CMBL using identical conditions

• Inhibition Studies:

  • Test sensitivity to chemical inhibitors that affect human CMBL

  • Determine IC50 values for each inhibitor

  • Compare inhibition profiles between sll1298 and human CMBL

• Structural Determinants of Specificity:

  • Conduct molecular docking simulations with identified substrates

  • Create chimeric enzymes by swapping domains between sll1298 and human CMBL

  • Perform site-directed mutagenesis of residues predicted to interact with substrates

Comparative Substrate Specificity Table:

Human CMBL has been shown to convert prodrugs such as olmesartan medoxomil, faropenem medoxomil, and lenampicillin to their active forms . By comparing the activity of sll1298 against these same substrates, researchers can determine functional conservation between bacterial and human homologs.

What are the best methods for studying the expression and localization of sll1298 in Synechocystis cells?

To investigate the expression and localization of sll1298 in Synechocystis sp. PCC 6803:

• Transcriptional Analysis:

  • RT-qPCR to quantify sll1298 mRNA levels under different conditions

  • RNA-seq to place sll1298 in the context of the global transcriptome

  • 5'-RACE to identify the transcription start site and promoter elements

  • Northern blotting to confirm transcript size and potential processing

• Protein Detection:

  • Generate specific antibodies against purified recombinant sll1298

  • Western blotting of cellular fractions (cytosol, membrane, periplasm, extracellular)

  • Create translational fusions with reporter tags (GFP, FLAG, His)

  • Quantify protein levels under varying growth conditions and stress factors

• Subcellular Localization:

  • Confocal microscopy of GFP-tagged sll1298

  • Immunogold electron microscopy with anti-sll1298 antibodies

  • Subcellular fractionation followed by activity assays and Western blotting

  • Analyze signal peptides and sorting sequences with bioinformatic tools

• Environmental Regulation:

  • Monitor expression under different light intensities and spectral qualities

  • Test effects of nutrient limitation (nitrogen, phosphorus, carbon)

  • Evaluate expression during different growth phases

  • Assess impact of stress conditions (oxidative, osmotic, temperature)

Fractionation Analysis Table:

The exoproteome analysis of Synechocystis sp. PCC 6803 could provide valuable insights regarding whether sll1298 is secreted or retained within the cell . Proteins in the exoproteome typically contain signal peptides recognized by the Sec system, involving proteins like SecA (sll0616) and SecY (sll1814) in Synechocystis .

How can CRISPR-based techniques be applied to study the function of sll1298 in Synechocystis?

CRISPR-based techniques offer powerful approaches for studying sll1298 function in Synechocystis:

• Gene Knockout Strategies:

  • Design sgRNAs targeting sll1298 coding sequence

  • Use CRISPR-Cas9 for precise gene disruption

  • Create markerless deletions to avoid polar effects

  • Generate complementation strains to confirm phenotypes

  • Analyze growth, metabolism, and stress resistance of knockout strains

• CRISPRi for Gene Repression:

  • Employ dCas12a-based CRISPRi system optimized for Synechocystis

  • Design gRNAs targeting the promoter or coding region of sll1298

  • Create an inducible knockdown system for temporal control

  • Quantify varying levels of repression with different gRNA designs

  • Monitor phenotypic effects under partial repression conditions

• CRISPRa for Overexpression:

  • Utilize the dCas12a-SoxS fusion system for targeted gene activation

  • Target gRNAs to the region 100-200 bp upstream of the transcription start site

  • Design gRNAs for the non-template strand for enhanced activation

  • Achieve controlled overexpression using the rhamnose-inducible promoter system

  • Quantify expression levels and resulting phenotypes

• Base Editing and Prime Editing:

  • Introduce specific mutations in catalytic residues without double-strand breaks

  • Create strains with altered substrate specificity or activity levels

  • Engineer tagged versions of the native protein for localization studies

  • Introduce regulatory sequence modifications to alter expression patterns

CRISPR Design Considerations Table:

The CRISPR activation system developed for Synechocystis offers a flexible editing window with minimal differences between gRNAs targeting the optimal region approximately -100 to -200bp upstream of the transcription start site . Notably, this system shows increased activation when targeting the non-template strand opposite the promoter direction .

What metabolic pathways in Synechocystis might involve sll1298, and how can this be experimentally verified?

To identify and verify the metabolic pathways involving sll1298:

• Bioinformatic Prediction Approaches:

  • Conduct genome context analysis (neighboring genes, operons)

  • Perform metabolic pathway reconstruction based on KEGG and BioCyc databases

  • Identify co-regulated genes through transcriptomic data mining

  • Search for conserved regulatory elements in the promoter region

• Metabolomic Analysis:

  • Compare metabolite profiles between wild-type and sll1298 knockout strains

  • Use untargeted LC-MS/MS to identify accumulated substrates or depleted products

  • Perform stable isotope labeling to track carbon flux through potential pathways

  • Focus on ester-containing metabolites based on predicted hydrolase activity

• Protein-Protein Interaction Studies:

  • Conduct pull-down assays with tagged sll1298

  • Perform bacterial two-hybrid screening

  • Use crosslinking and mass spectrometry (XL-MS) to identify interaction partners

  • Verify interactions with co-immunoprecipitation and bioluminescence resonance energy transfer (BRET)

• Synthetic Biology Validation:

  • Express sll1298 in heterologous hosts lacking the native enzyme

  • Reconstruct proposed pathways in vitro with purified components

  • Create synthetic operons containing sll1298 and predicted pathway genes

  • Use metabolic engineering to redirect flux through the pathway of interest

Potential Metabolic Roles Table:

Based on the metabolic engineering work done with Synechocystis sp. PCC 6803 for 1,2-propanediol production , researchers should consider whether sll1298 might interact with introduced heterologous pathways or affect native carbon flux. The Calvin-Benson-Bassham cycle in photosynthetic cyanobacteria generates various intermediates that could potentially be substrates for carboxymethylenebutenolidase activity.

How does the structure of sll1298 compare to human CMBL, and what are the implications for catalytic mechanism?

To compare the structure of sll1298 with human CMBL and elucidate catalytic mechanisms:

• Structural Analysis Approaches:

  • Generate homology models of sll1298 based on human CMBL structure

  • Identify conserved catalytic residues and substrate-binding pockets

  • Compare predicted secondary and tertiary structures

  • If possible, solve the crystal structure of recombinant sll1298

• Catalytic Mechanism Investigation:

  • Analyze conservation of the critical Cys132 residue from human CMBL

  • Perform site-directed mutagenesis of predicted catalytic residues

  • Measure pH-rate profiles to identify ionizable groups in catalysis

  • Conduct isotope exchange experiments to elucidate reaction intermediates

• Substrate Binding Studies:

  • Perform molecular docking simulations with potential substrates

  • Measure binding affinities using isothermal titration calorimetry (ITC)

  • Identify substrate-binding residues through hydrogen-deuterium exchange MS

  • Create variants with altered substrate specificity through rational design

• Evolutionary Comparison:

  • Conduct phylogenetic analysis of carboxymethylenebutenolidase homologs

  • Compare prokaryotic vs. eukaryotic enzyme family members

  • Identify conserved structural elements across evolutionary distance

  • Reconstruct the ancestral enzyme sequence and function

Structure-Function Relationship Table:

Human CMBL exhibits a unique sensitivity to chemical inhibitors that distinguishes it from other known esterases . Comparative inhibition studies between human CMBL and sll1298 would provide valuable insights into structural conservation of the active site. The site-directed mutagenesis approach used for human CMBL, where mutations C132A and C132S caused dramatic reduction in activity , provides a template for similar studies with sll1298.

Can sll1298 be engineered for enhanced catalytic efficiency or altered substrate specificity?

Engineering sll1298 for enhanced performance requires a systematic approach:

• Rational Design Strategies:

  • Identify catalytic bottlenecks through kinetic and structural analyses

  • Engineer the substrate-binding pocket based on homology models

  • Modify residues involved in rate-limiting steps

  • Introduce stabilizing mutations to improve thermostability

  • Design substrate tunnels for improved access to the active site

• Directed Evolution Approaches:

  • Develop high-throughput screening assays for desired properties

  • Create mutant libraries through error-prone PCR or DNA shuffling

  • Screen for variants with enhanced activity or altered substrate preference

  • Combine beneficial mutations through iterative rounds of evolution

  • Use computational tools to guide library design

• Structure-Guided Protein Engineering:

  • Utilize homology models or crystal structures to guide mutagenesis

  • Create chimeric enzymes with domains from related hydrolases

  • Introduce disulfide bridges for enhanced stability

  • Modify surface charges to improve solubility

  • Engineer allosteric regulation sites

• Expression Optimization:

  • Codon optimization for enhanced expression in Synechocystis

  • Test different promoter strengths and regulatory elements

  • Evaluate chromosomal integration versus plasmid-based expression

  • Compare expression at different genomic loci

Engineering Strategies Comparison Table:

Site-directed mutagenesis targeting the equivalent of Cys132 in human CMBL would be an excellent starting point for rational engineering. Integration of the engineered gene into different genomic sites should be considered, as the chromosomal location can impact expression levels and stability . The choice between chromosomal integration and plasmid-based expression would depend on the desired expression level and stability requirements .

How does sll1298 expression change under different environmental conditions, and what regulatory mechanisms control its expression?

To investigate the regulation of sll1298 expression:

• Transcriptional Profiling Approaches:

  • RNA-seq analysis under various growth conditions

  • Microarray profiling across environmental stress conditions

  • Targeted RT-qPCR of sll1298 under specific stimuli

  • Time-course experiments to capture dynamic expression patterns

  • Compare expression in wild-type vs. regulatory mutant strains

• Promoter Analysis:

  • Identify transcription start sites using 5'-RACE

  • Create promoter-reporter fusions with varying promoter lengths

  • Conduct site-directed mutagenesis of predicted regulatory elements

  • Perform electrophoretic mobility shift assays (EMSAs) to identify DNA-binding proteins

  • Use chromatin immunoprecipitation (ChIP) to verify regulatory interactions in vivo

• Post-Transcriptional Regulation:

  • Analyze mRNA stability under different conditions

  • Identify potential regulatory small RNAs targeting sll1298

  • Investigate RNA-binding proteins that may regulate translation

  • Examine the role of riboswitches or other RNA structural elements

• Environmental Response Testing:

  • Light intensity and spectral quality variations

  • Nutrient limitation (nitrogen, phosphorus, carbon)

  • Temperature shifts and heat shock response

  • Oxidative stress conditions

  • Exposure to potential substrates or pathway intermediates

Expression Regulation Table:

The expression of genes in Synechocystis often exhibits a narrow dynamic range under stress conditions, as observed in transcriptomic analyses . This characteristic should be considered when designing experiments to detect changes in sll1298 expression. The rhamnose-inducible promoter system (Prha) described for CRISPR applications could be valuable for controlled expression studies of sll1298.

What role might sll1298 play in cyanobacterial metabolic engineering and biotechnology applications?

Exploring the biotechnological potential of sll1298:

• Biocatalysis Applications:

  • Evaluate sll1298 for prodrug activation in pharmaceutical processes

  • Test activity against environmentally harmful esters for bioremediation

  • Develop immobilized enzyme formats for continuous bioprocessing

  • Engineer variants with desired properties for industrial applications

• Metabolic Engineering Integration:

  • Incorporate sll1298 into synthetic metabolic pathways

  • Use as a bioactivating enzyme for precursor conversion in biosynthetic pathways

  • Modulate native pathways by controlling intermediates through hydrolysis

  • Integrate with established engineering platforms for biofuel production

• Biosensor Development:

  • Create reporter systems based on sll1298 activity for detecting specific compounds

  • Develop whole-cell biosensors with sll1298-regulated promoters

  • Design enzyme-coupled assays for environmental monitoring

  • Create structure-based biosensors using protein engineering

• Synergy with Existing Platforms:

  • Integrate with the CRISPR activation system developed for Synechocystis

  • Combine with 1,2-propanediol production pathways

  • Evaluate interactions with the Calvin-Benson-Bassham cycle intermediates

  • Assess potential for exoproteome engineering

Biotechnology Applications Table:

Metabolic engineering of Synechocystis has already been demonstrated for the production of 1,2-propanediol, with yields reaching 950 mg/L . Understanding how sll1298 interacts with engineered pathways could help optimize production systems. The tolerance of Synechocystis cultures to compounds like 1,2-propanediol (up to 3% without growth effects) suggests that engineered strains could potentially accumulate significant amounts of target products.

What are the current limitations in our understanding of sll1298 and what research directions should be prioritized?

Current limitations and future research priorities for sll1298 studies:

Current Knowledge Gaps:

• Limited structural information about sll1298 compared to human CMBL

• Unclear physiological substrates and metabolic roles in Synechocystis

• Unknown regulatory mechanisms controlling expression

• Limited understanding of interactions with other cellular components

• Unexplored biotechnological potential

Priority Research Directions:

• Fundamental Characterization:

  • Solve the crystal structure of sll1298 to enable rational engineering

  • Identify natural substrates through metabolomics of knockout strains

  • Map the complete transcriptional regulation network

  • Determine subcellular localization and potential protein interactions

• Methodological Development:

  • Establish high-throughput screening systems for directed evolution

  • Develop specific activity assays for natural substrates

  • Create biosensor systems based on sll1298 activity

  • Optimize expression and purification protocols for structural studies

• Applied Research:

  • Evaluate potential for biodegradation of environmental contaminants

  • Test applications in pharmaceutical compound modification

  • Integrate into synthetic biology platforms for chemical production

  • Develop enzyme variants with enhanced stability for industrial applications

• Systems Biology Integration:

  • Model the role of sll1298 in the context of cyanobacterial metabolism

  • Integrate with genome-scale models of Synechocystis

  • Analyze evolutionary conservation across cyanobacterial species

  • Study the impact of environmental changes on expression patterns

The CRISPR activation system for Synechocystis provides a valuable tool for future studies, offering a flexible editing window and the ability to target the non-template strand for enhanced activation . The metabolic engineering strategies demonstrated for 1,2-propanediol production provide a framework for incorporating sll1298 into synthetic pathways. The characterization of the exoproteome offers insights into protein secretion mechanisms that might be relevant for sll1298 localization studies.