Recombinant Methanococcus maripaludis Aspartate carbamoyltransferase regulatory chain (pyrI)

What is the basic structure and function of aspartate carbamoyltransferase in M. maripaludis?

Aspartate carbamoyltransferase (ATCase) in M. maripaludis, like in other organisms, catalyzes the first committed step in pyrimidine biosynthesis - the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate. Studies on the related archaeon Methanococcus jannaschii have shown that the ATCase enzyme comprises two types of subunits: the catalytic chain (pyrB) that forms a trimer, and the regulatory chain (pyrI) that forms a dimer . This structural arrangement is similar to that observed in the well-characterized E. coli ATCase, although with notable differences in regulatory properties .

The catalytic function resides in the pyrB gene product, while the pyrI gene product is involved in the regulation of enzymatic activity. Research indicates that unlike the E. coli enzyme, the M. jannaschii ATCase (and by extension, the closely related M. maripaludis enzyme) exhibits limited homotropic cooperativity and less pronounced regulatory properties, suggesting adaptations specific to the archaeal metabolism .

How does the pyrI regulatory chain differ between M. maripaludis and other well-studied organisms?

The pyrI regulatory chain of M. maripaludis differs from that of other well-studied organisms such as E. coli in several significant ways. While both form multimeric structures, the M. maripaludis (and related M. jannaschii) pyrI forms dimers rather than the trimers seen in the catalytic subunit . In E. coli, the regulatory chain plays a crucial role in allosteric regulation, with CTP acting as a negative regulator and ATP as a positive regulator.

Studies with M. jannaschii ATCase suggest that the archaeal enzyme exhibits limited homotropic cooperativity and reduced regulatory properties compared to its E. coli counterpart . This difference likely reflects the adaptation of methanogenic archaea to their unique ecological niches and metabolic requirements. Homology modeling based on the three-dimensional structures of E. coli proteins has helped elucidate the structural basis for these functional differences, focusing on residues critical for catalysis, regulation, and quaternary structure formation .

What expression systems are typically used for recombinant production of M. maripaludis pyrI?

The recombinant production of M. maripaludis pyrI typically employs E. coli as an expression host due to its ease of genetic manipulation, rapid growth, and high protein yield. As demonstrated with the related M. jannaschii pyrI, these archaeal genes can be expressed at high levels in E. coli systems . The process generally involves:

• Codon optimization for E. coli expression if necessary

• Cloning the pyrI gene into appropriate expression vectors

• Transformation into E. coli expression strains

• Induction of protein expression

• Purification using affinity chromatography and other techniques

For genetic manipulation of M. maripaludis itself, researchers have developed several tools including a markerless mutagenesis method that uses the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase. This approach takes advantage of the ability to confer sensitivity to the base analog 8-azahypoxanthine . Additionally, recent advances include CRISPR/Cas12a-based genome-editing tools specifically adapted for methanogens including M. maripaludis .

What are the key considerations for designing experiments to characterize the regulatory properties of recombinant M. maripaludis pyrI?

When designing experiments to characterize the regulatory properties of recombinant M. maripaludis pyrI, researchers should consider a multi-faceted approach that accounts for the unique properties of archaeal proteins. Key considerations include:

• Stability and Folding: Ensure proper folding of the recombinant protein by optimizing expression conditions (temperature, induction time, media composition). Consider using archaeal chaperones if misfolding occurs.

• Assembly State Analysis: Employ techniques such as size exclusion chromatography, analytical ultracentrifugation, or native PAGE to confirm the dimeric state of pyrI, as observed in M. jannaschii . Compare with the trimeric state of pyrB to understand quaternary structure formation.

• Interaction Studies: Design experiments to investigate interactions between pyrI and pyrB subunits using techniques such as surface plasmon resonance, isothermal titration calorimetry, or co-immunoprecipitation.

• Regulatory Ligand Binding: Test the binding of potential regulatory nucleotides (ATP, CTP, UTP) using equilibrium dialysis or fluorescence-based assays, comparing results with the well-characterized E. coli system.

• Kinetic Analysis: Employ steady-state and pre-steady-state kinetics to characterize the regulatory effects on catalytic activity, particularly focusing on homotropic cooperativity which appears limited in the archaeal enzyme .

A Bayesian optimal experimental design (BOED) approach could be valuable for efficiently characterizing these regulatory properties, allowing researchers to maximize information gain from each experiment .

How can genetic manipulation techniques be optimized for studying pyrI function in M. maripaludis?

Optimizing genetic manipulation techniques for studying pyrI function in M. maripaludis requires specialized approaches suited to archaeal systems. Several strategies have proven effective:

• Markerless Mutagenesis: Utilize the established markerless mutagenesis system developed for M. maripaludis that employs the hpt gene encoding hypoxanthine phosphoribosyltransferase and the base analog 8-azahypoxanthine for negative selection . This approach allows for precise genomic modifications without leaving selection markers that might interfere with subsequent manipulations.

• CRISPR/Cas12a Systems: Implement the CRISPR/LbCas12a toolbox specifically adapted for methanogens. This system has been demonstrated as a reliable and quick method for genome editing in methanogenic archaea .

• Promoter Selection: Consider the relative strengths of various promoters when designing expression constructs. Strong constitutive promoters in M. maripaludis include PglnA, Pmtr, Pmcr, Pmcr_JJ, and Pfla_JJ . The choice of promoter should be informed by whether constitutive or regulated expression is desired.

• Growth Substrate Considerations: Be aware that promoter activity may vary depending on growth substrates (e.g., formate vs. H₂/CO₂), which can significantly impact gene expression levels .

• Complementation Strategies: For functional studies, consider both knockout approaches and complementation with wild-type or mutated versions of the pyrI gene at the upt locus, as demonstrated for other genes in M. maripaludis .

What mechanistic insights can be gained from comparative analysis of ATCase regulatory chains across archaeal and bacterial domains?

Comparative analysis of ATCase regulatory chains across archaeal and bacterial domains offers significant mechanistic insights into enzyme evolution, domain-specific adaptations, and regulatory mechanisms:

What are the optimal conditions for expressing recombinant M. maripaludis pyrI in heterologous systems?

The optimal conditions for expressing recombinant M. maripaludis pyrI in heterologous systems require careful consideration of multiple parameters:

For purification, a protocol similar to that established for M. jannaschii PyrI has been shown to be effective, typically involving affinity chromatography followed by size exclusion chromatography to isolate properly folded dimeric species . It's important to confirm the functional state of the purified protein through activity assays and structural characterization to ensure it reflects the native state.

How can researchers design experiments to study the interaction between pyrI and pyrB subunits?

Designing experiments to study the interaction between pyrI and pyrB subunits of M. maripaludis ATCase requires multiple complementary approaches:

• Co-expression Systems:

  • Design constructs for co-expression of both subunits with different affinity tags

  • Utilize polycistronic vectors or dual-plasmid systems with compatible origins of replication

  • Optimize expression conditions to ensure proper folding and assembly

• Interaction Analysis Techniques:

  • Surface Plasmon Resonance (SPR): Immobilize one subunit and measure binding kinetics of the other

  • Isothermal Titration Calorimetry (ITC): Quantify thermodynamic parameters of binding

  • Analytical Ultracentrifugation (AUC): Characterize assembly states and binding stoichiometry

  • FRET-based assays: Label subunits with appropriate fluorophores to monitor interaction in solution

• Structural Approaches:

  • X-ray crystallography of co-crystallized complexes

  • Cryo-EM for visualization of the holoenzyme structure

  • Cross-linking coupled with mass spectrometry to identify interaction interfaces

• Functional Assays:

  • Activity assays with varying ratios of pyrI to pyrB to determine optimal stoichiometry

  • Analysis of kinetic parameters in the presence and absence of regulatory subunits

  • Evaluation of regulatory effects of nucleotides on the assembled complex versus catalytic subunits alone

• Mutagenesis Studies:

  • Targeted mutations at the predicted interface based on homology models with E. coli ATCase

  • Alanine-scanning mutagenesis of conserved residues in the interaction interface

  • Charge-reversal mutations to disrupt salt bridges at the interface

When interpreting results, it's important to consider that the archaeal enzyme may exhibit different assembly properties and regulatory mechanisms compared to the well-characterized E. coli system, particularly given the observed limited homotropic cooperativity in M. jannaschii ATCase .

What methodological approaches are most effective for analyzing the regulatory properties of M. maripaludis ATCase?

Analyzing the regulatory properties of M. maripaludis ATCase requires sophisticated methodological approaches that can detect subtle changes in enzyme behavior under varying conditions:

• Steady-State Kinetics:

  • Spectrophotometric continuous assays monitoring product formation

  • Analysis of substrate-velocity curves to detect deviation from Michaelis-Menten kinetics

  • Determination of kinetic parameters (Km, Vmax, Hill coefficient) at varying substrate concentrations

  • Comparison of kinetic parameters in the presence of potential regulatory nucleotides (ATP, CTP, UTP)

• Ligand Binding Studies:

  • Equilibrium dialysis to measure binding of regulatory nucleotides

  • Fluorescence-based assays using intrinsic tryptophan fluorescence or extrinsic probes

  • Isothermal titration calorimetry to determine thermodynamic parameters of ligand binding

  • Surface plasmon resonance to measure binding kinetics

• Structural Dynamics Analysis:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to monitor conformational changes upon ligand binding

  • Small-angle X-ray scattering (SAXS) to detect quaternary structure changes in solution

  • Nuclear magnetic resonance (NMR) for detecting localized structural changes upon ligand binding

• Computational Approaches:

  • Molecular dynamics simulations to predict conformational changes upon ligand binding

  • Homology modeling based on E. coli structures to identify critical residues for regulation

  • Machine learning approaches to correlate sequence variations with regulatory properties

• Thermal Stability Assays:

  • Differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) to assess stabilization by regulatory ligands

  • Analysis of activity at different temperatures with and without regulatory ligands

Given the reported limited homotropic cooperativity and reduced regulatory properties of the related M. jannaschii ATCase , it's crucial to design experiments with sufficient sensitivity to detect subtle regulatory effects. Bayesian optimal experimental design (BOED) approaches can help maximize information gain from these experiments by identifying the most informative experimental conditions .

How should researchers analyze data from ATCase activity assays to identify subtle regulatory effects?

Analyzing data from ATCase activity assays to identify subtle regulatory effects requires sophisticated approaches, especially for archaeal enzymes like M. maripaludis ATCase that exhibit limited cooperative behavior compared to bacterial counterparts :

• Curve Fitting Beyond Michaelis-Menten:

  • Apply the Hill equation to quantify cooperativity:
    $v = \frac{V_{max}[S]^n}{K_{0.5}^n + [S]^n}$

  • Use the Monod-Wyman-Changeux (MWC) model to distinguish between cooperative mechanisms:
    $v = \frac{V_{max}(1+c\alpha)^{n-1}(\alpha+c\alpha^2)}{L(1+c\alpha)^n + (1+\alpha)^n}$
    where α = [S]/Ks, c = ratio of substrate affinity in R vs. T state, and L = equilibrium constant between R and T states.

• Statistical Analysis:

  • Employ global fitting of multiple datasets simultaneously to constrain parameters

  • Use Bayesian statistical approaches to quantify uncertainty in parameter estimates

  • Apply bootstrap resampling to estimate confidence intervals on kinetic parameters

  • Perform ANOVA or mixed-effects models to analyze data from multiple experimental conditions

• Visualization Techniques:

  • Create Eadie-Hofstee or Hanes-Woolf plots to visualize deviations from hyperbolic kinetics

  • Use heat maps to visualize the effects of multiple variables (substrate, effectors, pH)

  • Generate contour plots to identify optimal conditions for regulatory effects

• Comparative Analysis:

  • Normalize data relative to the unregulated enzyme to highlight subtle changes

  • Calculate activation or inhibition percentages at different effector concentrations

  • Compare kinetic parameters across related enzymes to identify archaeal-specific patterns

• Integrated Analysis:

  • Correlate kinetic data with structural information from homology models

  • Integrate binding data from ITC or fluorescence studies with activity measurements

  • Develop mathematical models that integrate multiple types of experimental data

When interpreting results, consider that subtle regulatory effects might be physiologically significant in the native context, even if they appear minimal compared to the pronounced effects seen in E. coli ATCase. The limited homotropic cooperativity observed in M. jannaschii ATCase suggests that archaeal enzymes may employ different regulatory strategies optimized for their metabolic context.

What approaches can help differentiate between experimental artifacts and genuine properties of recombinant M. maripaludis pyrI?

Differentiating between experimental artifacts and genuine properties of recombinant M. maripaludis pyrI requires rigorous controls and multiple complementary approaches:

• Multiple Expression and Purification Strategies:

  • Compare proteins purified under different conditions (detergents, salt concentrations, pH)

  • Test different purification tags (N-terminal vs. C-terminal, different tag types)

  • Evaluate tag-free protein obtained through proteolytic removal of fusion tags

  • Assess whether observed properties persist across all preparation methods

• Comparative Analysis with Related Proteins:

  • Compare with properties of homologous proteins from related species (e.g., M. jannaschii pyrI)

  • Test whether observed properties align with evolutionary expectations

  • Examine if structure-function relationships are conserved across related proteins

• Native vs. Recombinant Comparison:

  • When possible, isolate native enzyme from M. maripaludis for direct comparison

  • Compare properties of reconstituted holoenzyme with those reported for enzymes in cell extracts

  • Assess whether post-translational modifications might be responsible for differences

• Orthogonal Experimental Approaches:

  • Confirm key findings using multiple independent techniques

  • For structural determinations, compare results from different methods (X-ray, NMR, SAXS)

  • For functional assessments, use multiple activity assay formats

• Sensitivity Analysis:

  • Systematically vary experimental conditions to determine the robustness of observed properties

  • Test the effects of buffer components, stabilizing agents, and storage conditions

  • Evaluate time-dependent changes in protein properties to identify potential degradation effects

• Molecular Dynamics Simulations:

  • Use computational approaches to predict expected properties based on structure

  • Compare experimental results with simulation predictions

  • Identify potential non-physiological conformations that might arise during expression/purification

Researchers should be particularly alert to artifacts that might arise from expression in heterologous hosts, as archaeal proteins may adopt non-native conformations or aggregation states when produced in bacterial systems. The dimeric state observed for M. jannaschii pyrI should be confirmed for M. maripaludis pyrI using multiple independent methods.

How can structural modeling inform our understanding of M. maripaludis pyrI function?

Structural modeling approaches provide valuable insights into M. maripaludis pyrI function by connecting sequence information to three-dimensional structure and functional mechanisms:

• Homology Modeling Based on Related Structures:

  • Generate models based on the experimentally determined structures of homologous proteins, particularly E. coli pyrI and M. jannaschii pyrI if available

  • Validate models using energy minimization, Ramachandran plot analysis, and QMEAN scores

  • Compare models generated by different algorithms (SWISS-MODEL, I-TASSER, AlphaFold) to identify consistent structural features

• Functional Site Identification:

  • Analyze the conservation of residues at the interfaces between pyrI subunits in the predicted dimer

  • Identify potential regulatory nucleotide binding sites by comparison with known structures

  • Map conserved residues onto the structural model to identify functionally important regions

  • Examine the pyrI-pyrB interface to understand subunit interactions in the holoenzyme

• Molecular Dynamics Simulations:

  • Perform molecular dynamics simulations to assess structural stability and flexibility

  • Investigate conformational changes upon binding of regulatory nucleotides

  • Analyze water networks and ion binding sites that might be important for function

  • Simulate the effects of temperature on structure to understand thermoadaptation

• Electrostatic Surface Analysis:

  • Calculate surface electrostatic potentials to identify potential interaction sites

  • Compare electrostatic properties with those of homologous proteins from mesophilic organisms

  • Identify surface patches that might be involved in quaternary structure assembly

• In silico Mutagenesis:

  • Predict the effects of point mutations on structure and stability

  • Identify residues critical for maintaining the dimeric state observed in M. jannaschii pyrI

  • Design mutations that might alter regulatory properties for experimental testing

• Integration with Experimental Data:

  • Use structural models to interpret experimental results from mutagenesis studies

  • Design new experiments based on structural predictions

  • Refine models iteratively as new experimental data becomes available

As demonstrated with M. jannaschii pyrI, homology models constructed based on the three-dimensional structures of E. coli proteins can help identify residues critical for catalysis, regulation, and quaternary structure formation . These models are particularly valuable for understanding how the archaeal enzyme's unique properties emerge from its sequence and structure.

What are common challenges in purifying active recombinant M. maripaludis pyrI and how can they be addressed?

Purifying active recombinant M. maripaludis pyrI presents several challenges due to its archaeal origin and oligomeric nature. Here are common issues and their solutions:

A successful purification protocol for M. jannaschii PyrI has been reported , which can serve as a starting point for M. maripaludis pyrI purification. This protocol likely includes affinity chromatography followed by size exclusion chromatography to isolate the properly folded dimeric form. Confirming oligomeric state through analytical techniques such as size exclusion chromatography or analytical ultracentrifugation is crucial, as the expected dimeric structure of pyrI differs from the trimeric arrangement of pyrB .

How can researchers troubleshoot experiments involving pyrI-pyrB interactions?

Troubleshooting experiments involving pyrI-pyrB interactions requires systematic approaches to identify and address specific issues:

• No Detectable Interaction:

  • Verify protein activity and proper folding of individual components

  • Ensure buffer conditions are compatible with interaction (pH, salt concentration, divalent cations)

  • Try different protein concentrations, as archaeal ATCase may require different stoichiometry than E. coli

  • Add potential stabilizing ligands (substrates or regulatory nucleotides)

  • Consider tag position or removal if tags interfere with interaction sites

  • Use more sensitive detection methods such as SPR or fluorescence-based assays

• Non-specific Aggregation Instead of Proper Assembly:

  • Optimize buffer conditions to prevent aggregation (add glycerol, adjust salt concentration)

  • Try gradual dialysis to allow proper assembly

  • Add molecular crowding agents to mimic cellular conditions

  • Perform assembly at different temperatures

  • Use analytical ultracentrifugation to distinguish between specific complexes and aggregates

• Inconsistent Activity Measurements:

  • Standardize protein preparation methods

  • Ensure complete saturation of pyrB with pyrI by using excess pyrI

  • Control for time-dependent changes in activity

  • Verify that all necessary cofactors are present

  • Compare multiple activity assay formats to identify method-specific artifacts

• Inability to Detect Regulatory Effects:

  • Test wider concentration ranges of regulatory nucleotides

  • Consider that archaeal enzymes may respond to different effectors than E. coli

  • Ensure proper folding of the regulatory sites, which might require specific buffer conditions

  • Try combinations of potential regulatory molecules

  • Use more sensitive assays capable of detecting subtle changes in activity or conformation

• Difficulties in Structure Determination:

  • Try different crystallization conditions specific for protein complexes

  • Consider cross-linking to stabilize transient interactions

  • Use cryo-EM as an alternative approach for complex structures

  • Employ hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

Given that the M. jannaschii ATCase (closely related to M. maripaludis) exhibits limited homotropic cooperativity and reduced regulatory properties compared to E. coli , researchers should adjust their experimental approaches and expectations accordingly. The archaeal enzyme may have evolved different regulatory mechanisms adapted to its unique metabolic environment.

What strategies can improve the genetic manipulation of M. maripaludis for studying pyrI function in vivo?

Improving genetic manipulation of M. maripaludis for studying pyrI function in vivo requires specialized approaches tailored to this archaeal system:

• Optimized Selection Strategies:

  • Utilize the established markerless mutagenesis system based on the M. maripaludis hpt gene and 8-azahypoxanthine as a negative selectable marker

  • Implement the two-step recombination process: first integration of the entire plasmid, then counter-selection for the second recombination event

  • Consider the upt locus for stable incorporation of complementation constructs, as demonstrated for other genes

• CRISPR/Cas12a Implementation:

  • Adapt the CRISPR/LbCas12a toolbox developed for methanogens to target the pyrI gene

  • Design guide RNAs with high specificity for the pyrI target using methanogen-optimized algorithms

  • Optimize transformation protocols specifically for delivery of CRISPR components

• Expression Control:

  • Select appropriate promoters based on desired expression levels, with PglnA, Pmtr, Pmcr, Pmcr_JJ, and Pfla_JJ identified as strong promoters in M. maripaludis

  • Consider the effect of growth substrate on promoter activity, as some promoters show differential activity with formate versus H₂/CO₂

  • Use inducible promoters when available to control gene expression timing

• Transformation Enhancement:

  • Optimize polyethylene glycol (PEG)-mediated transformation protocols specifically for M. maripaludis

  • Consider the development of electroporation protocols if they improve transformation efficiency

  • Prepare DNA with appropriate methylation patterns to prevent restriction in M. maripaludis

• Phenotypic Analysis:

  • Develop high-throughput growth assays to detect subtle phenotypic effects of pyrI manipulation

  • Implement metabolomic approaches to monitor changes in pyrimidine metabolism

  • Consider synthetic lethal screens to identify genes that interact functionally with pyrI

• Complementation Strategies:

  • Design complementation constructs with varying levels of pyrI expression

  • Create point mutations in pyrI to test specific hypotheses about structure-function relationships

  • Use heterologous pyrI genes from related methanogens to test functional conservation

• Screening Approaches:

  • Develop screens based on pyrimidine analog sensitivity to identify functional pyrI variants

  • Implement fluorescence-based reporters when applicable for high-throughput analysis

  • Consider conditional lethal selections to identify functional mutants

These strategies build on established genetic tools for M. maripaludis, including the markerless mutagenesis system and the recently developed CRISPR/Cas12a tools for methanogens , while addressing the specific challenges of studying pyrI function in this archaeal system.