Recombinant Lactobacillus johnsonii CCA-adding enzyme (cca)

How are CCA-adding enzymes classified and what structural features define them?

CCA-adding enzymes are classified into two main categories: class I (found in archaea) and class II (found in eubacteria and eukaryotes). The classification is based on structural and sequence differences. Class I enzymes, like those from Sulfolobus shibatae, differ structurally from class II enzymes like those found in Escherichia coli and likely Lactobacillus johnsonii . Both classes catalyze the same reaction but employ different mechanisms for substrate recognition and nucleotide addition. The catalytic domain typically contains a conserved catalytic triad, and in some cases, specialized domains for nucleobase interaction.

What domains are typically found in bacterial CCA-adding enzymes?

Bacterial CCA-adding enzymes typically contain two critical domains: a catalytic head domain and a nucleobase-interacting neck domain. These domains work collaboratively to define nucleotide addition specificity and the number of nucleotides incorporated. In the class II CCA-adding enzyme from Thermotoga maritima, for instance, the head domain contains regions crucial for terminal A addition, while the neck domain contains amino acid residues (such as conserved Asp and Arg residues) that interact with nucleobases and define nucleotide incorporation specificity . A comparable domain organization would be expected in the L. johnsonii CCA-adding enzyme, including catalytic regions responsible for sequential CTP and ATP incorporation.

What expression systems are most effective for producing recombinant L. johnsonii CCA-adding enzyme?

For successful expression of recombinant L. johnsonii CCA-adding enzyme, E. coli-based expression systems are typically most effective. Similar to the approach used for the LJ0536 enzyme from L. johnsonii, a pET-based expression system in E. coli BL21(DE3) or a comparable strain would be appropriate . For optimal production, consider the following protocol:

• Clone the cca gene from L. johnsonii into a vector containing a strong inducible promoter (T7 or tac)

• Transform the construct into an expression strain optimized for protein production

• Grow cultures at 37°C until mid-log phase (OD600 ~0.6-0.8)

• Induce protein expression with IPTG (0.5-1.0 mM)

• Continue incubation at a reduced temperature (16-25°C) for 12-18 hours to enhance proper folding

This approach minimizes inclusion body formation while maximizing yield of functional enzyme. The specific growth conditions may require optimization depending on the particular properties of the L. johnsonii CCA-adding enzyme.

What purification strategy yields the highest activity for recombinant CCA-adding enzymes?

A multi-step purification approach is recommended to obtain highly active recombinant CCA-adding enzyme:

• Initial capture: Affinity chromatography using a His-tag or other suitable affinity tag is effective for initial purification. Include 5-10% glycerol and 1-2 mM DTT in all buffers to maintain enzyme stability.

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose) to remove contaminants with different charge properties.

• Polishing step: Size exclusion chromatography to remove aggregates and ensure homogeneity.

For specific activity preservation:

• Maintain pH between 7.0-8.0 throughout purification

• Include low concentrations (0.1-0.5 mM) of ATP or CTP in purification buffers

• Store the final preparation with 20-30% glycerol at -80°C in small aliquots to avoid freeze-thaw cycles

This strategy has been successful for purifying other nucleotidyltransferases and should be applicable to the L. johnsonii enzyme with minor modifications based on its specific properties.

What assays can be used to measure CCA-adding enzyme activity?

Several complementary approaches can be employed to measure the activity of recombinant L. johnsonii CCA-adding enzyme:

• Radioactive incorporation assay:

  • Incubate the enzyme with tRNA lacking CCA terminus and [α-32P]ATP or [α-32P]CTP

  • Measure incorporation of labeled nucleotides by scintillation counting or gel electrophoresis

  • This method is highly sensitive but requires radioactive material handling

• HPLC-based analysis:

  • Analyze the reaction products by ion-pair reverse-phase HPLC

  • Monitor the conversion of tRNA-N to tRNA-NC, tRNA-NCC, and tRNA-NCCA

  • Provides quantitative data on both CTP and ATP addition rates

• Gel-shift analysis:

  • Compare electrophoretic mobility of tRNA substrates before and after enzyme treatment

  • tRNAs with added nucleotides migrate differently, allowing for reaction monitoring

  • Can be enhanced with specific staining techniques or fluorescently labeled tRNAs

• Conditional amber suppression system:

  • In vivo functional assay where enzyme activity is linked to suppression of an amber codon

  • Similar to the method used to test chimeric CCA-adding enzymes

  • Provides information on the enzyme's ability to function in a cellular context

Each method has advantages for specific research questions, and combining multiple assays often provides the most comprehensive characterization.

How does nucleotide specificity develop in the CCA-adding reaction?

The nucleotide specificity in CCA-adding enzymes involves a sophisticated interplay between enzyme domains and the growing tRNA substrate. Based on structural studies of related enzymes:

• Template-independent selection: Unlike template-dependent polymerases, CCA-adding enzymes achieve nucleotide specificity without a nucleic acid template. The enzyme architecture creates specific binding pockets that select the correct nucleotide at each step.

• Dynamic remodeling: As each nucleotide is added, the enzyme-RNA complex undergoes conformational changes that reposition the 3' end of the tRNA and reshape the nucleotide binding pocket to select the next appropriate nucleotide.

• Domain collaboration: The head and neck domains work collaboratively to define both the number of nucleotide additions and the specificity for correct CCA addition . The neck domain contains amino acid residues that interact with nucleobases and prevent inappropriate nucleotide incorporation.

• β-turn involvement: A β-turn structure in the head domain (between β4 and β5) is involved in recognizing the 3' end of the tRNA during the CCA-adding reaction . This structural feature helps position the substrate correctly for each nucleotide addition.

In the L. johnsonii CCA-adding enzyme, similar mechanisms likely govern nucleotide selection, though specific residues involved may differ based on its unique sequence.

What factors affect the processivity of the CCA-adding enzyme?

Several factors influence the processivity of CCA-adding enzymes, which is crucial for complete and accurate CCA addition:

• Enzyme-tRNA interactions: Multiple contacts between the enzyme and the tRNA substrate maintain the tRNA in a fixed position during the consecutive addition of C and A nucleotides. This is evidenced by protection patterns of tRNA phosphates in both the acceptor stem and TPsiC stem-loop .

• Nucleotide concentration: Optimal processivity requires balanced concentrations of both CTP and ATP. Imbalanced nucleotide pools can lead to incomplete additions or misincorporation.

• Divalent metal ions: Mg2+ or Mn2+ ions are essential cofactors that stabilize the transition state during phosphodiester bond formation. Their concentration directly affects reaction rates and processivity.

• Flexible loop regions: Specific flexible loops in the catalytic domain define terminal A addition after CC addition . Mutations in these regions can disrupt the ordered addition process.

• Temperature and pH: The enzymatic activity and processivity of thermophilic versus mesophilic CCA-adding enzymes are differently affected by temperature and pH conditions, which must be optimized for the specific enzyme source.

For L. johnsonii CCA-adding enzyme, which comes from a mesophilic organism, optimal conditions likely include temperatures between 30-37°C and pH values near neutral (7.0-7.5).

What crystallization conditions are successful for bacterial CCA-adding enzymes?

Based on successful crystallization of related enzymes, the following conditions are recommended for crystallizing L. johnsonii CCA-adding enzyme:

• Protein preparation:

  • Highly purified enzyme (>95% purity by SDS-PAGE)

  • Concentrated to 5-15 mg/ml in a buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT

  • Removal of flexible tags that might interfere with crystal packing

• Crystallization screening:

  • Vapor diffusion methods (hanging or sitting drop)

  • Initial broad screening using commercial screens (Hampton, Qiagen, Molecular Dimensions)

  • Optimization focus on PEG-based conditions (PEG 3350-8000) with pH range 6.0-8.0

  • Addition of divalent cations (Mg2+, Mn2+) at 5-10 mM

• Co-crystallization with substrates:

  • For complex structures, include tRNA substrates (tRNA lacking CCA, tRNA-C, or tRNA-CC)

  • Alternatively, use catalytically inactive mutants (e.g., Ser→Ala in the catalytic triad)

  • Include nucleotide substrates (CTP or ATP) at 2-5 mM

• Cryoprotection:

  • Gradual transfer to mother liquor containing 20-25% glycerol, ethylene glycol, or PEG 400

  • Flash-cooling in liquid nitrogen

These conditions have proven successful for crystallizing the T. maritima CCA-adding enzyme and should provide a starting point for the L. johnsonii enzyme .

How can chimeric CCA-adding enzymes be designed to alter specificity or efficiency?

Design of chimeric CCA-adding enzymes provides valuable insights into structure-function relationships and can generate enzymes with novel properties. Based on successful domain swapping experiments:

• Identify conserved domains: Analyze sequence alignments to identify the catalytic head domain and nucleobase-interacting neck domain in your enzymes of interest.

• Design junction points: Select junction points at domain boundaries to minimize disruption of secondary structure elements. Successful chimeras have been created by exchanging entire domains between related enzymes, such as between T. maritima CCA-adding enzyme and A. aeolicus A-adding enzyme .

• Region-specific exchanges:

  • Head domain exchanges affect nucleotide specificity

  • Neck domain exchanges influence the number of nucleotide incorporations

  • β-turn regions in the head domain are involved in 3' end recognition

• Functional analysis strategy:

  Chimera Design	Expected Effect	Testing Method
  Head domain exchange	Altered terminal nucleotide specificity	In vitro nucleotide addition assay
  Neck domain exchange	Changed incorporation pattern	Gel-based analysis of products
  Both domains exchanged	Complete functional conversion	In vivo complementation assay
  β-turn modification	Altered RNA recognition	Binding affinity measurements

• Validation approaches: Test chimeric enzymes both in vitro using purified components and in vivo using conditional amber suppression systems or other functional complementation assays .

This approach can potentially create a hybrid enzyme with the specificity of one enzyme and the efficiency or thermal stability of another, opening possibilities for biotechnological applications.

How does the L. johnsonii CCA-adding enzyme compare to other bacterial homologs?

The L. johnsonii CCA-adding enzyme likely shares key features with other bacterial homologs but may possess unique characteristics based on its probiotic origin:

• Classification: As a bacterial enzyme, it falls into the class II category of CCA-adding enzymes, similar to those from E. coli and T. maritima .

• Comparative sequence analysis: Sequence alignments would reveal conservation of key catalytic residues observed in other bacterial enzymes:

  • The catalytic triad (likely Ser, His, and Asp residues)

  • Nucleobase-interacting residues in the neck domain (Asp and Arg)

  • β-turn motifs involved in 3' end recognition

• Thermal stability profile: Unlike enzymes from thermophiles like T. maritima, the L. johnsonii enzyme likely shows optimal activity at lower temperatures (30-37°C) reflecting its mesophilic origin.

• Specificity mechanisms: The collaborative action of head and neck domains in defining nucleotide selection is likely conserved, but subtle differences in amino acid composition may affect kinetic parameters.

• Insertions and deletions: Unique sequence features may exist, similar to how the cinnamoyl esterase from L. johnsonii (LJ0536) contains a unique inserted α/β subdomain that shapes its catalytic pocket .

A detailed phylogenetic analysis would place the L. johnsonii enzyme in context within the broader family of bacterial CCA-adding enzymes and highlight its potentially unique characteristics.

What site-directed mutagenesis studies would be most informative for understanding catalytic mechanism?

Based on structural and functional studies of related enzymes, the following site-directed mutagenesis studies would provide valuable insights into the catalytic mechanism of L. johnsonii CCA-adding enzyme:

• Catalytic triad mutations:

  • Identify and mutate the presumptive catalytic residues (Ser→Ala, His→Ala, Asp→Ala)

  • These mutations should completely abolish activity while preserving substrate binding

  • Create a catalytically inactive enzyme for co-crystallization studies with substrates

• Nucleobase-interacting residues:

  • Target conserved Asp and Arg residues in the neck domain

  • Mutations should affect nucleotide specificity and may result in misincorporation

• β-turn modifications:

  • Mutate residues in the β-turn that recognize the 3' end of the RNA

  • Evaluate effects on discrimination between different 3' end states (tRNA, tRNA-C, tRNA-CC)

• Recommended mutation design and analysis:

  Target	Mutation Type	Expected Effect	Analysis Method
  Catalytic Ser	Ser→Ala	Complete loss of activity	Activity assays
  Neck domain Asp/Arg	Asp→Asn, Arg→Lys	Altered nucleotide specificity	Product analysis by HPLC
  β-turn residues	Conservative substitutions	Changed 3' end recognition	Kinetic analysis with different substrates
  Interface residues	Ala scanning	Identify key tRNA contacts	Binding assays

• Interface residues: Create a set of surface mutations at the presumed tRNA binding interface to map critical contact points for substrate recognition.

These studies would build a comprehensive picture of structure-function relationships in the L. johnsonii CCA-adding enzyme and provide insights into the mechanistic details of nucleotide selection and addition.

What computational approaches can predict substrate interactions for the L. johnsonii CCA-adding enzyme?

Advanced computational methods can provide valuable insights into enzyme-substrate interactions for CCA-adding enzymes:

• Homology modeling:

  • Build a structural model of the L. johnsonii CCA-adding enzyme using crystal structures of related enzymes as templates

  • Refine the model using molecular dynamics simulations to optimize local geometry

  • Validate the model using structural assessment tools (PROCHECK, VERIFY3D)

• Molecular docking studies:

  • Dock tRNA substrates (tRNA, tRNA-C, tRNA-CC) into the enzyme model

  • Identify key residues involved in tRNA recognition and positioning

  • Dock nucleotide substrates (CTP, ATP) to predict binding modes at each stage

• Molecular dynamics simulations:

  • Perform extended simulations (>100 ns) of enzyme-substrate complexes

  • Analyze conformational changes during substrate binding and catalysis

  • Identify water networks and ion coordination essential for catalysis

• Quantum mechanical calculations:

  • Model the reaction mechanism at the QM or QM/MM level

  • Calculate energy barriers for nucleotide addition

  • Predict effects of mutations on transition state stabilization

• Sequence coevolution analysis:

  • Identify coevolving residues that may be functionally linked

  • Predict structural contacts not evident from static structures

  • Guide experimental design for double mutant cycles

These computational approaches, ideally performed in parallel with experimental validation, can provide mechanistic insights and guide rational enzyme engineering for improved activity or altered specificity.

What are the optimal reaction conditions for in vitro activity of recombinant L. johnsonii CCA-adding enzyme?

For maximal activity of recombinant L. johnsonii CCA-adding enzyme in vitro, the following conditions are recommended:

• Buffer composition:

  • 50 mM Tris-HCl or HEPES pH 7.5-8.0

  • 50-100 mM KCl or NaCl

  • 10 mM MgCl2 (critical for catalysis)

  • 1 mM DTT (to maintain reduced state of cysteine residues)

  • 0.1 mg/ml BSA (for enzyme stability)

• Nucleotide substrates:

  • CTP: 0.2-1.0 mM

  • ATP: 0.2-1.0 mM

  • Equal concentrations of both nucleotides for balanced incorporation

• Temperature and incubation time:

  • 30-37°C (optimal for mesophilic L. johnsonii)

  • 15-60 minutes depending on enzyme concentration and assay sensitivity

• tRNA substrate concentration:

  • 0.5-5 μM for kinetic studies

  • Higher concentrations may be used for preparative reactions

• Enzyme concentration:

  • 10-100 nM for kinetic studies

  • Higher concentrations (0.5-1 μM) for complete conversion in preparative reactions

These conditions should be optimized for the specific recombinant enzyme preparation, as minor variations in the enzyme construct (tags, fusion partners) can affect optimal reaction parameters.

How can the stability of recombinant CCA-adding enzyme be improved for long-term storage?

Ensuring long-term stability of recombinant CCA-adding enzyme is crucial for consistent experimental results. The following strategies are effective:

• Storage buffer optimization:

  • 20-50 mM Tris-HCl or HEPES pH 7.5

  • 100-200 mM KCl or NaCl

  • 1-2 mM DTT or 5 mM β-mercaptoethanol

  • 0.1-0.5 mM EDTA to sequester trace metals

  • 20-50% glycerol as cryoprotectant

• Aliquoting strategy:

  • Divide purified enzyme into small aliquots (10-50 μl)

  • Use screw-cap microcentrifuge tubes with O-rings to prevent evaporation

  • Store at -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles

• Stabilizing additives:

  • 0.1-0.5 mg/ml BSA as a carrier protein

  • 0.1% Triton X-100 or NP-40 (non-ionic detergents)

  • 0.1-0.5 mM ATP (substrate-induced stabilization)

• Alternative stabilization methods:

  • Lyophilization in the presence of stabilizers (trehalose, sucrose)

  • Immobilization on solid supports for repeated use

  • Engineering thermostable variants by comparing with thermophilic homologs

• Quality control:

  • Periodically test enzyme activity from stored aliquots

  • Monitor for signs of aggregation or proteolysis

  • Consider SEC analysis to confirm enzyme remains monomeric or in its native oligomeric state

These approaches can significantly extend the useful life of the enzyme preparation and ensure reproducible results across extended research projects.