Recombinant Sulfolobus solfataricus Putative esterase SSO2140 (SSO2140)

What is Sulfolobus solfataricus and why is the putative esterase SSO2140 significant for research?

Sulfolobus solfataricus is a hyperthermophilic crenarchaeon that thrives in extremely hot, acidic environments with optimal growth conditions around 80°C . This organism has become an important model system in archaeal biology due to its stable genome and established genetic tools .

The putative esterase SSO2140 belongs to a class of hydrolytic enzymes (esterases/lipases) found in Sulfolobus species that have significant biotechnological potential due to their exceptional stability under extreme conditions . These enzymes catalyze the hydrolysis of ester bonds and are valuable for various applications requiring operation under harsh conditions. The thermostability and potential catalytic versatility of SSO2140 make it particularly interesting for both fundamental research into archaeal biochemistry and potential biotechnological applications.

What expression systems are most effective for recombinant production of SSO2140?

For recombinant expression of SSO2140, researchers have successfully employed several systems:

• Homologous expression in Sulfolobus species: Using the virus vector-based pMJ0503 system, which has proven effective for the overexpression of tagged proteins in S. solfataricus . This approach maintains the native cellular environment for proper folding.

• Heterologous expression in S. acidocaldarius: The glucose ABC transporter system from S. solfataricus has been successfully expressed in S. acidocaldarius strain MW001, suggesting this could be a viable approach for SSO2140 as well .

• E. coli expression systems: While not explicitly mentioned for SSO2140 in the provided sources, E. coli systems are commonly used for archaeal proteins, though special considerations must be made for proteins from hyperthermophiles.

When expressing hyperthermophilic proteins in mesophilic hosts, consider codon optimization, using specialized E. coli strains with additional chaperones, and expression at lower temperatures to promote proper folding rather than inclusion body formation.

How can I verify the esterase activity of recombinant SSO2140?

To verify esterase activity of recombinant SSO2140, implement the following methodological approach:

• Spectrophotometric assays: Use p-nitrophenyl esters of varying chain lengths (C2-C16) as substrates. The release of p-nitrophenol can be monitored at 405-410 nm. Perform assays at elevated temperatures (60-85°C) to assess thermophilic activity.

• Temperature-activity profiling: Conduct activity assays across a temperature range (40-95°C) to determine the optimal temperature for enzymatic activity.

• pH profiling: Assess activity across a range of pH values (typically pH 2-10) using appropriate buffer systems stable at high temperatures.

• Substrate specificity analysis: Test activity against a panel of substrates with different chain lengths and structures to characterize the enzyme's preference.

• Inhibition studies: Use common esterase inhibitors (PMSF, EDTA) to confirm the catalytic mechanism.

For thermostable enzymes like SSO2140, ensure all buffers and reagents are thermostable, and consider using sealed reaction vessels to prevent evaporation during high-temperature incubation.

What are the optimal conditions for cultivating Sulfolobus solfataricus for SSO2140 studies?

For optimal cultivation of S. solfataricus in SSO2140 studies:

Growth conditions:

• Temperature: 80°C (optimal for S. solfataricus growth)

• pH: 2-3 (acidic conditions)

• Media: Use specific media containing appropriate carbon sources such as glucose, galactose, or arabinose

• Aeration: Maintain aerobic conditions as S. solfataricus has an aerobic respiratory chain

Carbon source considerations:
S. solfataricus metabolizes sugars through a branched Entner-Doudoroff (ED) pathway . When studying esterases like SSO2140, consider how different carbon sources might affect enzyme expression. The organism can utilize various sugars including glucose, galactose, and in the case of S. solfataricus (but not S. acidocaldarius), D-arabinose .

Monitoring growth:
Track growth by measuring optical density at 600 nm. Note that UV irradiation (if used in experiments) can cause growth arrest and cell lysis at high doses (>3300 J/m²) .

Growth arrest for synchronization:
If synchronized cultures are needed, controlled UV exposure can be used, as it causes temporary growth arrest in S. solfataricus similar to what occurs in E. coli .

How can I genetically manipulate Sulfolobus solfataricus to study SSO2140 function?

For genetic manipulation of S. solfataricus to study SSO2140:

Available genetic tools:

• Vector systems: Several plasmid vectors are available, including:

  • pNOB8 plasmid and SSV1 virus-based vectors

  • pMJ0503 virus vector (useful for tagged protein overexpression)

  • For S. islandicus, the pSeSD1 plasmid has proven effective

• Gene knockout approaches:

  • Markerless deletion mutants can be created using uracil auxotrophy systems

  • For S. acidocaldarius, use strain MW001 or MR31 (uracil auxotrophic mutants)

  • Construct gene deletion cassettes with flanking homologous regions

• Marker systems:

  • The β-galactosidase LacS can be used as a marker in strain PBL2025, which has a large deletion including sugar metabolism genes

  • Uracil auxotrophy is commonly used for selection and counterselection

Recommended approach for SSO2140 studies:

• For expression studies: Use the virus vector-based pMJ0503 for overexpression of tagged SSO2140

• For knockout studies: Consider S. acidocaldarius system due to its genomic stability compared to S. islandicus strains, which contain numerous transposable elements that may lead to genome rearrangements

• For functional complementation: After creating a knockout, reintroduce the SSO2140 gene (wild-type or mutated versions) to confirm phenotypes

Note that conditions for gene disruption by homologous recombination of exogenous DNA into the S. solfataricus genome have been established, facilitating genetic manipulation .

What purification strategies are most effective for obtaining active SSO2140?

To purify active SSO2140, implement a multi-step approach designed for thermostable proteins:

Initial purification steps:

• Heat treatment: Capitalize on SSO2140's thermostability by heating cell lysates (70-80°C for 10-30 minutes) to precipitate most E. coli proteins if using a heterologous expression system

• Ammonium sulfate fractionation: Use differential precipitation to remove contaminants

Chromatographic purification:
3. Immobilized metal affinity chromatography (IMAC): If using a His-tagged construct, perform at high temperatures (60°C) to maintain native conformation
4. Ion exchange chromatography: Select appropriate resin based on the theoretical pI of SSO2140
5. Hydrophobic interaction chromatography: Particularly useful for esterases due to their surface hydrophobicity
6. Size exclusion chromatography: As a final polishing step to achieve high purity

Considerations for thermostable enzymes:

• Use buffers with higher melting temperature salts (phosphate rather than Tris)

• Consider adding stabilizing agents (glycerol, specific metal ions)

• Perform activity assays at each purification step to track specific activity

• Store in glycerol-containing buffers at -20°C or -80°C for long-term stability

Activity preservation:

• Avoid repeated freeze-thaw cycles

• Test stability under various storage conditions (including room temperature)

• Consider lyophilization for long-term storage

This purification strategy leverages the inherent thermostability of SSO2140 to facilitate separation from mesophilic contaminant proteins.

How does SSO2140 compare structurally and functionally to other archaeal esterases?

SSO2140 belongs to a diverse family of carboxyl esterases found in archaea . While specific structural data for SSO2140 is limited in the provided sources, comparative analysis with other archaeal esterases reveals several common features:

Structural features of archaeal esterases:

• Typically contain the canonical α/β hydrolase fold

• Possess the characteristic catalytic triad (Ser-His-Asp/Glu)

• Often exhibit higher content of charged amino acids on the protein surface, contributing to thermostability

• May contain disulfide bridges and ion-pair networks that enhance structural rigidity

Functional comparison:

Archaeal esterases like SSO2140 often exhibit remarkable stability under conditions that would denature mesophilic enzymes, making them valuable subjects for both fundamental research and biotechnological applications.

What is known about SSO2140's catalytic mechanism and substrate specificity?

Based on the general characteristics of archaeal esterases and the metabolic context of S. solfataricus:

Expected catalytic mechanism:
SSO2140 likely employs the classical serine hydrolase mechanism involving:

• Nucleophilic attack by the active site serine on the carbonyl carbon of the ester substrate

• Formation of a tetrahedral intermediate

• Release of the alcohol component

• Hydrolysis of the acyl-enzyme intermediate

• Release of the acid component and regeneration of the enzyme

Substrate specificity considerations:

• As a putative esterase rather than a lipase, SSO2140 would typically prefer:

  • Short to medium chain esters (C2-C8)

  • Water-soluble substrates over insoluble ones

  • Carboxyl esters over other ester types

Experimental approach to determine specificity:

• Assay activity against p-nitrophenyl esters of varying chain lengths (C2-C16)

• Test natural substrates relevant to S. solfataricus metabolism

• Examine activity on structurally diverse esters to map the substrate binding pocket

• Conduct kinetic analyses (Km, kcat, kcat/Km) for preferred substrates

• Perform inhibition studies with various compounds to probe active site characteristics

Potential metabolic relevance:
Within S. solfataricus, SSO2140 may be involved in:

• Lipid metabolism

• Degradation of specific carbon sources

• Modification of cell envelope components

• Response to environmental stresses

Detailed biochemical characterization is necessary to confirm these predictions and establish SSO2140's precise substrate profile and biological role.

How does temperature affect the stability and activity of SSO2140?

As an enzyme from the hyperthermophilic archaeon S. solfataricus, SSO2140 exhibits remarkable thermal properties that are central to its research interest:

Temperature-activity relationship:

• Optimal temperature: Likely around 80-85°C, corresponding to S. solfataricus' optimal growth temperature

• Activity range: Expected to maintain significant activity between 65-95°C

• Cold inactivation: May exhibit reduced activity at temperatures below 50°C, a phenomenon observed in some hyperthermophilic enzymes

Thermal stability parameters:

• Half-life: Likely exhibits extended half-life (hours to days) at temperatures of 70-80°C

• Denaturation temperature (Tm): Expected to be above 90°C

• Irreversible denaturation: May only occur at temperatures approaching or exceeding 100°C

Molecular basis of thermostability:
S. solfataricus proteins, including SSO2140, achieve thermostability through several mechanisms:

• Increased number of ion-pair networks

• Higher proportion of charged amino acids on the surface

• Compact packing of hydrophobic core

• Reduced number of thermolabile amino acids (Asn, Gln, Cys, Met)

• Potential disulfide bridges in extracellular enzymes

Experimental considerations:
When studying SSO2140's thermal properties, researchers should:

• Use temperature-controlled spectrophotometers for accurate activity measurements

• Employ differential scanning calorimetry (DSC) to determine Tm values

• Conduct thermal inactivation studies at various temperatures to establish stability profiles

• Consider the stability of substrates at high temperatures when designing assays

The exceptional thermostability of SSO2140 not only provides insights into protein adaptation to extreme conditions but also makes it potentially valuable for high-temperature biotechnological applications.

What strategies can be employed to engineer SSO2140 for altered substrate specificity?

To engineer SSO2140 for modified substrate specificity, consider implementing these advanced approaches:

Rational design strategies:

• Structure-guided mutagenesis: If a crystal structure or reliable homology model is available, identify residues in the substrate-binding pocket and introduce mutations that alter pocket size, shape, or electrostatic properties

• Substrate docking simulations: Use computational modeling to predict how mutations might affect substrate binding

• Loop engineering: Modify loop regions that often determine substrate access and binding

• Catalytic residue modifications: Fine-tune the positioning of catalytic triad residues to accommodate different substrates

Directed evolution approaches:

• Error-prone PCR: Generate libraries with random mutations throughout the SSO2140 gene

• DNA shuffling: Recombine related esterase genes to create chimeric enzymes with novel properties

• High-throughput screening: Develop colorimetric or fluorescence-based assays compatible with the target substrates to screen large variant libraries

• Selection systems: Design growth-coupled selection systems in S. acidocaldarius or E. coli

Special considerations for thermostable enzymes:

• Engineer at temperatures below the optimal to identify variants that maintain thermostability

• Screen for activity at both high and moderate temperatures to ensure stability is preserved

• Consider the "stability buffer" - thermostable enzymes can often tolerate more destabilizing mutations

Potential targets for engineering:

• Converting esterase activity to lipase activity by enhancing binding of longer-chain substrates

• Engineering enantioselectivity for specific chiral compounds

• Modifying pH optima while maintaining thermostability

• Introducing new catalytic activities while preserving the thermostable scaffold

Successful engineering requires balancing the desired changes in specificity with the maintenance of thermostability and catalytic efficiency.

How can I study the role of SSO2140 in the context of S. solfataricus metabolism?

To investigate the metabolic role of SSO2140 in S. solfataricus, implement a multi-faceted systems biology approach:

Genetic approaches:

• Gene knockout: Create a markerless deletion of SSO2140 using established genetic tools for Sulfolobus species

• Conditional expression: Develop systems for controlled expression to study phenotypes under various conditions

• Reporter fusions: Create transcriptional or translational fusions to monitor expression patterns

Physiological characterization:

• Growth phenotyping: Compare growth of wild-type and mutant strains on various carbon sources

• Stress response analysis: Examine response to pH, temperature, or oxidative stress

• UV and DNA damage response: Assess whether SSO2140 expression changes during DNA damage response, as S. solfataricus has a sophisticated response to UV damage

Omics approaches:

• Transcriptomics: Analyze global transcriptional changes in knockout strains

• Proteomics: Identify protein interaction partners through co-immunoprecipitation or crosslinking studies

• Metabolomics: Identify metabolite changes in knockout strains to infer biochemical pathways affected

Biochemical analysis:

• In vivo substrate identification: Use metabolite profiling to identify naturally occurring substrates

• Enzyme localization: Determine subcellular localization using tagged versions of SSO2140

• Activity correlation: Correlate enzyme expression/activity with specific metabolic states

Contextual integration:
Consider how SSO2140 might function within known S. solfataricus metabolic pathways:

• Central carbohydrate metabolism via the branched Entner-Doudoroff pathway

• Lipid metabolism and membrane adaptation to extreme conditions

• Potential role in stress responses or adaptation to changing environments

• Possible involvement in the citric acid cycle or related pathways

This comprehensive approach will provide insights into the physiological role of SSO2140 beyond its biochemical characterization.

What are the challenges and solutions for obtaining structural data for SSO2140?

Obtaining high-quality structural data for SSO2140 presents several unique challenges related to its archaeal origin and thermophilic nature:

Challenges in structural studies:

• Protein production issues:

  • Expression in heterologous hosts may yield improperly folded protein

  • Aggregation during concentration steps

  • Post-translational modifications may differ from native host

• Crystallization difficulties:

  • High surface charge typical of thermophilic proteins can impede crystal formation

  • Conformational flexibility or domain movements may prevent crystallization

  • Requirement for specific cofactors or substrates for stable conformations

• Data collection considerations:

  • Radiation damage during X-ray diffraction

  • Phase determination challenges for novel structures

  • Need for high-resolution data to interpret thermostability features

Methodological solutions:

For protein production:

• Express in S. acidocaldarius using established vector systems

• Use specialized E. coli strains with additional chaperones

• Co-express with archaeal chaperones to assist proper folding

• Purify at elevated temperatures to promote native folding

For crystallization:

• Screen with substrate analogs or inhibitors to stabilize active site

• Use surface entropy reduction (SER) to replace surface charged clusters

• Try in situ proteolysis to remove flexible regions

• Explore lipidic cubic phase for membrane-associated forms

Alternative structural approaches:

• Cryo-electron microscopy (cryo-EM) for larger assemblies

• Small-angle X-ray scattering (SAXS) for solution structure determination

• Nuclear magnetic resonance (NMR) for dynamic regions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational states

Data analysis considerations:

• Molecular dynamics simulations at elevated temperatures to understand thermal adaptations

• Comparative analysis with mesophilic homologs to identify thermostability determinants

• Analysis of ion-pair networks and hydrophobic packing

Successful structural characterization of SSO2140 would provide valuable insights into both the catalytic mechanism of this archaeal esterase and the structural basis of extreme thermostability.

How does SSO2140 compare to recombinase paralogs from S. solfataricus?

While SSO2140 (a putative esterase) and recombinase paralogs like SsoRal3 represent entirely different protein families with distinct functions, comparing their research contexts provides valuable insights:

Functional comparison:

Research methodology similarities:
Both proteins can be studied using similar experimental approaches:

• Heterologous expression and purification

• Biochemical characterization of substrate specificity

• Structure-function relationship analysis

• In vivo knockout studies to determine physiological roles

Insights from recombinase research applicable to SSO2140:
SsoRal3 has been biochemically characterized as a ssDNA-dependent ATPase that catalyzes strand invasion and influences the activity of the main recombinase SsoRadA . This work demonstrates:

• The importance of studying archaeal enzyme paralogs to understand their specialized functions

• The value of both in vitro biochemical studies and interaction studies with related proteins

• Successful approaches for expressing and purifying functional archaeal proteins

Researchers working with SSO2140 might consider similar analytical approaches to define its interactions with other metabolic enzymes and its specific role within esterase families in S. solfataricus.

What DNA damage response mechanisms in S. solfataricus might affect SSO2140 expression?

S. solfataricus exhibits sophisticated DNA damage response systems that could potentially influence SSO2140 expression:

Known DNA damage response in S. solfataricus:

• UV-induced DNA damage triggers growth arrest and transcriptional changes

• Cyclobutane pyrimidine dimers (CPDs) are efficiently repaired in vivo in the dark, suggesting an active nucleotide excision repair (NER) pathway

• UV exposure induces transcription of NER genes XPF, XPG, and XPB

• DNA damage response includes regulation of genes encoding DNA binding proteins involved in chromosome dynamics

• Several genes are induced by both UV irradiation and the intercalating agent actinomycin D

Potential implications for SSO2140:

• Transcriptional regulation: If SSO2140 is involved in stress response or DNA damage repair pathways, its expression might be co-regulated with known DNA repair genes

• Functional connections: SSO2140 could potentially play a role in:

  • Modifying damaged DNA ends

  • Processing lipid peroxidation products resulting from oxidative stress

  • Restructuring cell membrane components during stress response

• Experimental approach to investigate potential connections:

  • Analyze SSO2140 promoter for DNA damage response elements

  • Perform quantitative RT-PCR to assess SSO2140 expression following UV exposure

  • Compare expression patterns with known DNA repair genes like XPF, XPG, and XPB

  • Investigate potential physical interactions with DNA repair proteins through co-immunoprecipitation

Research design considerations:

• Use similar UV exposure protocols (200-3300 J/m²) as described for S. solfataricus DNA damage studies

• Monitor growth recovery patterns after DNA damage in wild-type versus SSO2140 knockout strains

• Consider the polycistronic nature of many S. solfataricus transcripts when analyzing expression data

This research direction would explore potential non-canonical functions of SSO2140 beyond its predicted esterase activity, potentially revealing unexpected roles in stress response pathways.

How might SSO2140 be integrated into synthetic biology applications requiring thermostable enzymes?

SSO2140, as a thermostable esterase from S. solfataricus, presents numerous opportunities for integration into synthetic biology frameworks requiring robust enzymatic components:

Potential synthetic biology applications:

• Thermophilic metabolic engineering:

  • Integration into synthetic pathways for biofuel production at elevated temperatures

  • Development of consolidated bioprocessing systems operating at high temperatures

  • Construction of minimal archaeal chassis organisms with defined enzymatic capabilities

• Enzyme cascade systems:

  • Coupling with other thermostable enzymes for multi-step biocatalytic processes

  • Creation of immobilized enzyme reactors for continuous processing

  • Development of self-assembling protein scaffolds incorporating multiple thermostable enzymes

• Biosensor technologies:

  • Design of thermostable biosensors for environmental monitoring in harsh conditions

  • Development of field-deployable detection systems with extended shelf-life

  • Integration into microfluidic devices for high-temperature operations

Design considerations for synthetic biology applications:

Implementation strategies:

• Characterize SSO2140 parts (promoters, coding sequence, terminators) according to synthetic biology standards

• Develop orthogonal expression systems compatible with both mesophilic and thermophilic hosts

• Create libraries of SSO2140 variants with modified properties for different applications

• Establish high-throughput screening systems to evaluate performance in synthetic contexts

The exceptional stability of thermophilic enzymes like SSO2140 makes them particularly valuable for synthetic biology applications requiring robust performance under challenging conditions, extended operational lifetimes, and resistance to chemical denaturants.