Recombinant Gloeobacter violaceus Phosphoribosylformylglycinamidine synthase 2 (purL), partial

What is the function of PurL in purine biosynthesis?

PurL, also known as formylglycinamide ribonucleotide amidotransferase (FGAR-AT), catalyzes the fourth step in the purine biosynthetic pathway. The enzyme specifically converts formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, inorganic phosphate, and glutamate . This reaction represents a crucial amidation step in the de novo purine synthesis pathway, making PurL essential for nucleotide formation. The reaction mechanism involves ATP-mediated activation of the FGAR amide moiety, followed by an amide transfer from glutamine, which serves as the ammonia source in the reaction .

The enzymatic action of PurL can be monitored using coupling enzymes such as PurM and through modified Bratton-Marshall assays that detect the formation of FGAM. The protein's activity is typically assayed in buffers containing HEPES, potassium and magnesium chloride, with ATP and glutamine as substrates .

Why is Gloeobacter violaceus significant in evolutionary studies?

Gloeobacter violaceus holds particular significance in evolutionary studies as it represents one of the earliest-diverging cyanobacterial lineages. G. violaceus PCC 7421 is distinctive among cyanobacteria for its lack of thylakoid membranes, which are specialized internal membrane structures present in virtually all other photosynthetic cyanobacteria . This characteristic suggests that G. violaceus diverged before the evolution of thylakoid membranes in the cyanobacterial lineage.

Phylogenetic analyses consistently place Gloeobacterales as an early-branching group in cyanobacterial evolution, forming a distinct clade along with other early-diverging groups like Thermostichales . Proteins from G. violaceus, including PurL, often exhibit unique features that reflect this evolutionary position. Studying these proteins provides valuable insights into the ancestral state of cyanobacterial metabolism and how metabolic pathways evolved over billions of years of cyanobacterial evolution.

What structural forms of PurL exist across bacterial species?

Two distinct structural forms of PurL have been characterized across bacterial species:

The large PurL contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with an ammonia channel located between the active sites of the latter two domains . The small PurL is structurally homologous to just the FGAM synthetase domain of large PurL and must form a complex with two additional proteins, PurQ (containing glutaminase activity) and PurS (homologous to the N-terminal domain of large PurL) .

What are optimal expression systems for recombinant G. violaceus PurL production?

Expression of recombinant G. violaceus PurL requires careful optimization of both the expression system and conditions. While I haven't found specific data for G. violaceus PurL in the search results, I can provide methodological guidance based on successful expression of other cyanobacterial proteins and PurL from other organisms:

When expressing recombinant G. violaceus PurL, researchers should:

• Consider using a tag system (His6, GST, etc.) for purification while ensuring the tag doesn't interfere with the protein's structure or function.

• Test expression at different temperatures (15-37°C) as cyanobacterial proteins often express better at lower temperatures.

• Optimize induction conditions, including inducer concentration and induction time.

• Test solubility enhancement strategies such as co-expression with chaperones if inclusion body formation occurs.

The optimal expression system may differ depending on whether you're working with full-length or partial PurL constructs, as partial constructs may have different folding requirements.

How can Design of Experiments (DoE) be applied to optimize recombinant G. violaceus PurL expression?

Design of Experiments (DoE) provides a powerful approach to optimize recombinant protein expression, including G. violaceus PurL. Unlike the inefficient one-factor-at-a-time method, DoE considers the combined effects of multiple factors simultaneously, producing more reliable results with fewer experiments .

A methodological approach to applying DoE for G. violaceus PurL expression would involve:

• Factor Identification: Identify critical factors affecting expression such as temperature, inducer concentration, media composition, induction time, cell density at induction, and post-induction duration.

• Experimental Design Selection: Choose an appropriate DoE method:

  • Factorial designs for screening significant factors

  • Response surface methodology (RSM) for optimizing conditions

  • Central composite or Box-Behnken designs for detailed optimization

• Response Variables Definition: Define clear measurable outcomes such as:

  • Protein yield (mg/L culture)

  • Soluble fraction percentage

  • Specific activity

  • Purity after initial capture step

• Experimental Execution and Analysis: Use available software packages to:

  • Design the experimental runs

  • Analyze results using statistical methods

  • Generate predictive models

  • Identify optimal conditions

By systematically exploring these factors through DoE, researchers can identify optimal conditions with a significantly reduced number of experiments compared to traditional approaches .

What purification strategies yield highest activity for G. violaceus PurL?

Purification of recombinant G. violaceus PurL requires strategies that preserve both structural integrity and enzymatic activity. Based on successful purification of other PurL proteins described in the literature , the following methodological approach is recommended:

• Initial Capture:

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

  • Consider buffer conditions that maintain protein stability (typically 50 mM HEPES pH 7.2-7.5, 20-50 mM KCl, 20 mM MgCl2)

  • Include stabilizing agents such as glycerol (5-10%) and potentially reducing agents (DTT or β-mercaptoethanol)

• Intermediate Purification:

  • Ion exchange chromatography (typically anion exchange as most PurL proteins have acidic pI)

  • Optimize salt gradient for elution to separate PurL from contaminating proteins

• Polishing:

  • Size exclusion chromatography to obtain homogeneous protein preparation

  • Useful for assessing oligomeric state and removing aggregates

• Activity Preservation Considerations:

  • Include cofactors like magnesium ions in purification buffers

  • Consider adding ATP or ATP analogs to stabilize the protein

  • If using tag cleavage, assess activity before and after to ensure function is maintained

For partial PurL constructs, particular attention should be paid to protein stability, as truncated proteins may have exposed hydrophobic patches that promote aggregation. Consider adding stabilizing agents such as arginine or additional salt if aggregation occurs.

How does the structure of G. violaceus PurL compare to PurL complexes from other organisms?

While the specific structure of G. violaceus PurL is not directly described in the search results, a comparative structural analysis can be inferred based on information about PurL proteins from other organisms :

G. violaceus, as a Gram-negative cyanobacterium, likely contains the large form of PurL consisting of a single polypeptide chain with three distinct domains. This structure would contrast with the PurLQS complex found in Gram-positive bacteria like Bacillus subtilis and archaea like Thermotoga maritima .

The T. maritima PurLQS complex structure reveals that complex formation is dependent on glutamine and ADP, suggesting these metabolites mediate the recruitment of PurQ and PurS . The conformational changes observed upon complex formation elucidate the mechanism of complex assembly and activation .

In G. violaceus large PurL, similar conformational changes would likely occur within the single polypeptide in response to substrate binding. The ammonia channel, critical for transferring ammonia from the glutaminase domain to the FGAM synthetase domain, would be formed within the protein rather than at protein-protein interfaces as in the PurLQS complex.

What methods are most effective for resolving contradictory data in G. violaceus PurL structural studies?

When facing contradictory data in structural studies of G. violaceus PurL, researchers should employ multiple complementary approaches to resolve discrepancies:

• Integrated Structural Biology Approach:

  • Combine X-ray crystallography, cryo-EM, NMR, and small-angle X-ray scattering (SAXS)

  • Each method has strengths and limitations; agreement across methods increases confidence

  • Use molecular dynamics simulations to investigate dynamic aspects not captured in static structures

• Functional Validation:

  • Perform site-directed mutagenesis of key residues identified in structural models

  • Correlate structural features with kinetic parameters

  • Use chemical crosslinking combined with mass spectrometry to validate domain interactions

• Comparative Analysis:

  • Compare with structures of homologous proteins

  • Analyze conservation patterns of key residues across species

  • Consider phylogenetic context when interpreting structural data

• Address Experimental Artifacts:

  • Verify protein oligomeric state in solution versus crystal

  • Check for effects of purification tags on structure

  • Consider buffer conditions that might affect conformational states

By systematically addressing contradictions through multiple methods and careful experimental design, researchers can develop a more accurate and comprehensive understanding of G. violaceus PurL structure and function.

How can evolutionary analysis of G. violaceus PurL inform functional studies?

The evolutionary significance of G. violaceus as an early-diverging cyanobacterium makes its PurL protein particularly valuable for understanding the evolution of purine biosynthesis. Methodological approaches to leverage evolutionary insights include:

• Phylogenetic Reconstruction:

  • Construct phylogenetic trees of PurL sequences across diverse bacterial phyla

  • Use Maximum Likelihood, Bayesian, and distance-based methods to ensure robust topology

  • Recover G/T/HC topology (Gloeobacterales/Thermostichales/Higher Crown) as observed in other conserved proteins

  • Analyze without outgroups to test robustness of tree topology

• Ancestral Sequence Reconstruction:

  • Infer ancestral PurL sequences at key nodes in bacterial evolution

  • Express and characterize these reconstructed ancient enzymes

  • Compare kinetic parameters between ancestral and extant enzymes

• Conserved Domain Analysis:

  • Identify highly conserved residues across all PurL proteins

  • Distinguish between conservation patterns in large versus small PurL forms

  • Map conservation onto structural models to identify functional hotspots

• Horizontal Gene Transfer (HGT) Assessment:

  • Analyze gene neighborhoods surrounding purL in different genomes

  • Look for incongruence between PurL phylogeny and species phylogeny

  • Consider the impact of HGT on functional adaptations of PurL

The evolutionary analysis can reveal how substrate specificity, catalytic efficiency, and regulatory mechanisms have evolved over time. Insights from comparing G. violaceus PurL with homologs from diverse bacteria can guide the design of mutations to alter enzyme properties or engineer novel functions.

By integrating evolutionary analysis with structural and functional studies, researchers can develop a more comprehensive understanding of how this essential enzyme has evolved and adapted throughout bacterial history.

What are the optimal assay conditions for measuring G. violaceus PurL activity?

Measuring the enzymatic activity of G. violaceus PurL requires careful optimization of assay conditions. Based on established methods for PurL from other organisms , researchers should consider the following methodological approach:

• Basic Assay Components:

  • Buffer: 50 mM HEPES pH 7.2

  • Salt: 20 mM KCl

  • Divalent cation: 20 mM MgCl₂

  • Substrates: 10 mM ATP, 1 mM β-FGAR, 20 mM L-glutamine

• Detection Methods:

  • Coupled enzyme assay using E. coli PurM as coupling enzyme

  • Modified Bratton-Marshall assay for FGAM detection

  • ADP production monitoring via pyruvate kinase/lactate dehydrogenase coupling

• Optimization Considerations:

  • Temperature range (25-45°C for cyanobacterial enzymes)

  • pH optimization (typically 7.0-8.0)

  • Magnesium:ATP ratio (usually 2:1)

  • Substrate concentration ranges for kinetic parameter determination

For partial PurL constructs, it may be necessary to supplement the assay with additional components if the construct lacks certain functional domains. If working with a construct analogous to small PurL, consider adding purified PurQ and PurS proteins to reconstitute activity.

Given the ancestral nature of G. violaceus, researchers should also test alternative nitrogen donors beyond glutamine, as the enzyme might exhibit interesting substrate preferences that reflect its evolutionary position.

How do post-translational modifications affect G. violaceus PurL activity?

While specific information about post-translational modifications (PTMs) of G. violaceus PurL is not provided in the search results, this important research question can be approached methodologically:

• Identification of Potential PTMs:

  • Mass spectrometry analysis of purified native and recombinant PurL

  • Comparison between protein expressed in different systems (E. coli, yeast, insect cells)

  • Western blotting with PTM-specific antibodies (phospho-, glyco-, acetyl-specific)

• Common PTMs to Investigate:

  • Phosphorylation: Often regulates enzyme activity in response to cellular energy status

  • Acetylation: May regulate catalytic activity or protein-protein interactions

  • Methylation: Can affect protein stability and interactions

  • Oxidation of cysteine residues: May regulate activity in response to redox conditions

• Functional Characterization:

  • Compare kinetic parameters of modified versus unmodified protein

  • Create site-directed mutants that mimic or prevent specific PTMs

  • Test activity under different cellular conditions that might affect PTM status

• Evolutionary Context:

  • Compare PTM sites across cyanobacterial species

  • Analyze conservation of PTM sites in the context of PurL evolution

  • Consider whether PTMs represent ancient regulatory mechanisms or more recent adaptations

Given G. violaceus's evolutionary position as an early-diverging cyanobacterium, analysis of its PurL PTMs could provide insights into the ancient regulatory mechanisms of purine biosynthesis and how these have evolved in more complex photosynthetic organisms.

What are common challenges in expressing partial PurL constructs from G. violaceus?

Expression of partial PurL constructs from G. violaceus presents several challenges that researchers should anticipate. While specific information about G. violaceus PurL expression is not directly provided in the search results, methodological approaches to address these challenges can be outlined:

• Domain Boundary Selection:

  • Improper domain boundary selection can lead to misfolded, insoluble protein

  • Use multiple bioinformatic tools (InterPro, PFAM, CATH) to predict domain boundaries

  • Design several constructs with varying boundaries (+/- 5-10 residues)

  • Include natural linker regions when possible

• Solubility Issues:

  • Partial constructs often expose hydrophobic regions normally buried in the full protein

  • Test multiple solubility-enhancing fusion tags (MBP, SUMO, Trx, GST)

  • Optimize induction conditions (lower temperature, reduced inducer concentration)

  • Screen various buffer additives (arginine, glycerol, non-detergent sulfobetaines)

• Structural Integrity:

  • Partial constructs may lack stabilizing interactions from missing domains

  • Conduct thermal shift assays to assess stability of different constructs

  • Consider co-expression with interacting partners (e.g., for small PurL constructs, co-express with PurQ and PurS)

  • Use circular dichroism to verify secondary structure content

• Functional Assays for Partial Constructs:

  • Partial constructs may lack complete catalytic activity

  • Design domain-specific functional assays (e.g., ATP binding for synthetase domain)

  • Consider reconstitution experiments by mixing separately purified domains

  • Use biophysical methods to assess substrate binding if catalytic activity is absent

Understanding the interdomain interactions in the full-length PurL is crucial for successful expression of functional partial constructs. The challenges encountered may themselves provide insights into the structural organization and functional interdependence of the domains in G. violaceus PurL.

How can contradictory results in PurL phylogenetic studies be resolved?

Resolving contradictory results in phylogenetic studies of PurL requires a systematic methodological approach that addresses various sources of uncertainty:

• Sequence Selection and Alignment Quality:

  • Ensure comprehensive taxon sampling across bacterial diversity

  • Use multiple sequence alignment methods (MUSCLE, MAFFT, T-Coffee)

  • Apply alignment trimming tools to remove poorly aligned regions

  • Test the impact of including/excluding specific sequences or taxonomic groups

• Phylogenetic Method Selection:

  • Apply multiple phylogenetic inference methods (Maximum Likelihood, Bayesian, Neighbor-Joining)

  • Test various evolutionary models and select the best-fit model using AIC/BIC criteria

  • Conduct topology tests to statistically evaluate alternative tree hypotheses

  • Assess the impact of using site-heterogeneous models (e.g., CAT, CAT-GTR)

• Addressing Specific Challenges:

  • Long-branch attraction: Use slow-fast method to identify fast-evolving sites

  • Compositional bias: Apply models that account for compositional heterogeneity

  • Horizontal gene transfer: Compare gene and species trees, use reconciliation methods

  • Paralogy issues: Carefully distinguish between large PurL and small PurL sequences

• Integration with Other Evidence:

  • Compare PurL phylogeny with phylogenies of other purine biosynthesis genes

  • Incorporate genomic context information (gene order, operon structure)

  • Consider structural constraints in interpreting evolutionary patterns

  • Evaluate results in light of known major evolutionary transitions

What strategies can overcome protein stability issues with G. violaceus PurL?

Protein stability is a common challenge when working with recombinant proteins, particularly those from organisms with unique environmental adaptations like G. violaceus. While specific stability data for G. violaceus PurL is not provided in the search results, a methodological approach to addressing stability issues includes:

• Buffer Optimization Through Design of Experiments (DoE):

  • Apply DoE principles to systematically explore buffer components

  • Test ranges of pH (6.0-9.0), salt concentration (0-500 mM), and buffer types

  • Evaluate stabilizing additives (glycerol, arginine, sucrose, polyols)

  • Screen for stabilizing effects of substrates and cofactors (ATP, FGAR, glutamine)

• Thermal Stability Assessment and Enhancement:

  • Use differential scanning fluorimetry (Thermofluor) to screen stabilizing conditions

  • Conduct melting curve analysis under different buffer conditions

  • Test cryoprotectants for storage stability (glycerol, ethylene glycol)

  • Evaluate the effects of oxidation (add reducing agents like DTT or TCEP)

• Protein Engineering Approaches:

  • Identify flexible regions using hydrogen-deuterium exchange mass spectrometry

  • Introduce stabilizing mutations based on homology to thermostable homologs

  • Consider surface entropy reduction to enhance crystallizability

  • Design stabilizing disulfide bonds in regions showing high B-factors

• Formulation for Long-term Stability:

  • Optimize protein concentration (dilute vs. concentrated storage)

  • Determine optimal storage temperature (-80°C, -20°C, 4°C)

  • Test lyophilization conditions and reconstitution buffers

  • Evaluate stabilizers like trehalose, sucrose, or bovine serum albumin

By applying these methodological approaches through a systematic design of experiments, researchers can significantly improve the stability of recombinant G. violaceus PurL, enhancing the reproducibility and reliability of subsequent structural and functional studies.