Recombinant Methanococcus maripaludis Phosphoribosylglycinamide formyltransferase 2 (purT)

What is Methanococcus maripaludis and why is it suitable for purT expression studies?

Methanococcus maripaludis is a genetically tractable, mesophilic, hydrogenotrophic methanogen with a fully sequenced genome containing 1,722 protein-coding genes. It represents an excellent model organism for studying archaeal metabolism due to its rapid growth rate, well-established genetic tools, and relatively simple cultivation requirements .

M. maripaludis is particularly valuable for heterologous protein expression studies because:

• It possesses genome-scale metabolic models that facilitate metabolic engineering approaches

• It has established systems for both large-scale and continuous cultivation

• It can utilize various carbon sources including CO2 and formate

• Its genetic tractability allows for precise genome manipulation and protein expression

These characteristics make M. maripaludis an ideal chassis for expressing and studying proteins involved in purine biosynthesis pathways, including Phosphoribosylglycinamide formyltransferase 2 (purT).

How does the phosphate-regulated expression system function in M. maripaludis?

The phosphate-regulated expression system in M. maripaludis is based on the pst promoter, which responds to inorganic phosphate concentration in the growth medium. This system offers several key advantages for recombinant protein expression:

• Gene expression increases 4- to 6-fold when medium phosphate drops to growth-limiting concentrations

• The system decouples growth from heterologous gene expression without requiring addition of exogenous inducers

• Expression typically initiates between mid-log phase and early stationary phase, reducing potential toxicity effects of recombinant proteins

• The minimal pst promoter contains a conserved AT-rich region, a factor B recognition element (BRE), and a TATA box for phosphate-dependent regulation

This regulated expression system has demonstrated significant improvements in expressing potentially toxic proteins compared to constitutive promoters like the histone promoter (PhmvA). In particular, it has successfully expressed the methyl-coenzyme M reductase complex and the arginine methyltransferase MmpX, which were difficult to express using other systems .

What are the critical parameters for optimizing purT expression using the phosphate-regulated system?

Optimizing purT expression in M. maripaludis using the phosphate-regulated system requires careful consideration of several parameters:

Notably, rational changes to the factor B recognition element and start codon have been shown to have minimal impact on expression levels, while modifications to the transcription start site and 5' untranslated region can significantly affect protein production while maintaining phosphate-dependent regulation .

How can expression levels of recombinant purT be quantitatively assessed?

Quantitative assessment of recombinant purT expression levels requires a multi-faceted approach:

• Protein Quantification: Western blotting with antibodies against purT or epitope tags (such as FLAG or His-tag) provides direct visualization and quantification of protein levels. Densitometric analysis of band intensity enables semi-quantitative comparison across samples.

• Enzymatic Activity Assays: Spectrophotometric assays measuring the formylation of phosphoribosylglycinamide (GAR) to formylglycinamide ribonucleotide (FGAR) offer functional quantification of active enzyme.

• mRNA Quantification: Reverse transcription quantitative PCR (RT-qPCR) allows measurement of purT transcript levels, providing insights into transcriptional regulation.

• Mass Spectrometry: Targeted proteomics approaches such as selected reaction monitoring (SRM) can provide absolute quantification of purT protein when appropriate internal standards are employed.

For comparative studies, expression should be normalized to cell density and assessed at multiple time points following phosphate limitation to establish expression kinetics and identify optimal harvest times.

What computational approaches are most effective for predicting purT structure and function?

Multiple computational approaches can be employed for predicting the structure and function of M. maripaludis purT:

• Homology Modeling: AlphaFold2 and RoseTTAFold provide accurate structural predictions by leveraging homology to known structures of purT from related organisms.

• Molecular Dynamics Simulations: GROMACS or AMBER can model protein flexibility and substrate interactions in different solution conditions, providing insights into the enzyme's conformational dynamics.

• Substrate Docking: AutoDock Vina or HADDOCK can predict binding modes of phosphoribosylglycinamide and formyl donors, identifying key residues involved in substrate recognition.

• Phylogenetic Analysis: Comparative genomics approaches can identify conserved motifs across methanogenic archaea, highlighting functionally important regions.

• Metabolic Network Analysis: Integration of purT function within genome-scale metabolic models of M. maripaludis can predict the system-level impact of purT modulation.

These computational predictions should be validated experimentally through site-directed mutagenesis of predicted catalytic residues and substrate binding sites, followed by kinetic characterization of the mutant enzymes.

How do the kinetic properties of M. maripaludis purT compare to those from other organisms?

While specific kinetic data for M. maripaludis purT is not provided in the search results, a comparative analysis framework should include:

The kinetic characterization should account for the mesophilic nature of M. maripaludis compared to thermophilic methanogens like Methanocaldococcus jannaschii, which may result in distinct temperature and pH optima, as well as potential differences in substrate specificity and catalytic efficiency .

What are the most effective strategies for purifying active recombinant purT from M. maripaludis?

Purification of active recombinant purT from M. maripaludis requires specialized approaches that account for the unique properties of archaeal proteins:

• Expression System Selection:

  • The phosphate-regulated pst promoter system offers significant advantages over constitutive promoters for potentially toxic proteins

  • C-terminal or N-terminal FLAG-tagging has been successfully employed for recombinant protein detection and purification in M. maripaludis

• Cell Lysis Optimization:

  • Anaerobic conditions should be maintained during cell disruption to preserve enzyme activity

  • Gentle lysis methods (e.g., osmotic shock or detergent-based approaches) may better preserve protein structure than mechanical disruption

• Chromatography Sequence:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Anti-FLAG affinity chromatography for FLAG-tagged constructs

  • Ion exchange chromatography to separate based on charge differences

  • Size exclusion chromatography as a final polishing step

• Activity Preservation:

  • Addition of stabilizing agents such as glycerol (10-20%) to all buffers

  • Inclusion of reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues in reduced state

  • Potential supplementation with substrate analogs or product to stabilize active conformation

Each purification step should be validated by assessing both protein purity (SDS-PAGE) and specific activity (enzyme assay) to ensure that purification procedures maintain the functional integrity of the enzyme.

How can isotope labeling be used to elucidate the catalytic mechanism of purT?

Isotope labeling approaches provide powerful tools for investigating the catalytic mechanism of purT:

• 13C-Labeled Substrates: Using 13C-labeled formate or formyl donors allows tracking of carbon transfer during the formylation reaction, confirming the reaction mechanism and identifying any potential intermediates.

• 18O-Labeled Water: Incorporation studies with H218O can determine if water molecules participate in the reaction mechanism, particularly in potential hydrolysis steps.

• 15N-Labeled Phosphoribosylglycinamide: Enables monitoring of nitrogen-containing groups during catalysis, helping to elucidate transition states.

• 2H-Labeled Substrates: Kinetic isotope effect studies using deuterated substrates can identify rate-limiting steps in the catalytic mechanism.

• NMR Spectroscopy: Analysis of labeled substrates and products using 13C, 15N, and 31P NMR can provide detailed information about bond formation and cleavage during catalysis.

The incorporation of isotope labeling with mass spectrometry and NMR spectroscopy offers a comprehensive approach to resolving reaction mechanisms, particularly in distinguishing between potential alternative pathways for formyl group transfer.

How does purT function integrate with the broader metabolic network of M. maripaludis?

The purT enzyme in M. maripaludis functions within a complex metabolic network that connects purine biosynthesis with central carbon metabolism and energy generation:

• Purine Biosynthesis Pathway: purT catalyzes the formylation of phosphoribosylglycinamide to formylglycinamide ribonucleotide, a critical step in de novo purine biosynthesis.

• One-Carbon Metabolism: The enzyme utilizes formyl donors that integrate with the broader one-carbon metabolic network, potentially connecting to methanogenesis pathways unique to M. maripaludis.

• Energy Metabolism: The ATP-dependent nature of purT links its activity to the energy status of the cell, making it responsive to the unique energy conservation mechanisms of methanogens.

• Nitrogen Metabolism: M. maripaludis can utilize free nitrogen as its sole nitrogen source, which may influence the availability of nitrogen-containing precursors for purine biosynthesis .

Understanding these metabolic interconnections is crucial when considering purT as a target for metabolic engineering, as alterations in its activity may have ripple effects throughout the metabolic network of M. maripaludis.

What are the methodological approaches for investigating purT function in vivo?

Investigating purT function within living M. maripaludis cells requires specialized approaches:

• Gene Deletion/Complementation: Creating purT deletion mutants followed by complementation with variant forms of the enzyme can establish the phenotypic consequences of altered purT function.

• Conditional Expression Systems: The phosphate-regulated pst promoter system provides an excellent platform for controlled expression of purT variants, allowing temporal control over enzyme levels .

• Metabolomics: Targeted and untargeted metabolomic analyses can identify changes in purine intermediates and related metabolites in response to purT modulation.

• Flux Analysis: 13C metabolic flux analysis can trace carbon flow through the purine biosynthesis pathway, quantifying the impact of purT alterations on metabolic flux distributions.

• Growth Phenotyping: Systematic assessment of growth characteristics under varying nutrient conditions can reveal the physiological impact of purT modifications.

• Transcriptomics/Proteomics: Multi-omics approaches can identify compensatory responses to purT modulation, revealing regulatory networks connected to purine metabolism.

These methodologies, when applied in combination, provide a systems-level understanding of purT function within the native cellular context of M. maripaludis.

How can researchers address common challenges in purT activity assays?

Researchers frequently encounter several challenges when performing purT activity assays:

When interpreting purT activity data, researchers should consider:

• The physiological context of M. maripaludis, including its optimal growth temperature and intracellular pH

• The potential impact of assay components on enzyme stability and activity

• The need for appropriate controls to account for non-enzymatic reactions and background signals

• The importance of biological replicates to establish statistical significance

How can researchers reconcile discrepancies between in vitro and in vivo purT activity measurements?

Discrepancies between in vitro biochemical assays and in vivo observations of purT activity are common and can be addressed through a systematic approach:

• Physiological Relevance of Assay Conditions:

  • Ensure in vitro buffer composition, pH, and ionic strength mirror the intracellular environment of M. maripaludis

  • Consider the impact of molecular crowding on enzyme kinetics by including crowding agents like PEG or Ficoll

• Substrate Availability and Concentrations:

  • Measure or estimate intracellular concentrations of phosphoribosylglycinamide and formyl donors

  • Adjust in vitro assay concentrations to reflect physiological ranges

• Post-translational Modifications:

  • Investigate potential modifications of purT in vivo that may not be present in recombinant systems

  • Consider the role of archaeal-specific modifications that might affect enzyme function

• Protein-Protein Interactions:

  • Explore potential interaction partners that might modulate purT activity in vivo

  • Perform pull-down experiments to identify proteins that co-purify with purT

• Metabolite Regulation:

  • Test the effect of other cellular metabolites on purT activity

  • Consider allosteric regulation mechanisms that might operate in vivo

• Transport and Compartmentalization:

  • Assess the localization of purT within M. maripaludis cells

  • Consider potential barriers to substrate accessibility in vivo

By systematically addressing these factors, researchers can develop more accurate models of purT function in its native cellular context.