Recombinant Mesoplasma florum Adenylosuccinate synthetase (purA)

What is the role of Adenylosuccinate synthetase (purA) in Mesoplasma florum metabolism?

Adenylosuccinate synthetase (purA) in M. florum catalyzes the first committed step in the de novo biosynthesis of AMP from IMP. Specifically, it forms adenylosuccinate from IMP and aspartate using GTP as an energy source. In the broader context of M. florum's minimal metabolism, purA plays a crucial role in purine nucleotide metabolism, which is essential for RNA and DNA synthesis.

While M. florum possesses a remarkably streamlined genome with only ~685 protein-coding sequences, purine metabolism pathways remain conserved, highlighting their fundamental importance . According to proteomic analyses, most M. florum proteins involved in purine and pyrimidine metabolism (approximately 23 proteins representing 3.4% of total ORFs) are expressed at detectable levels, demonstrating their essential role even in this near-minimal bacterium .

How does the genomic context of purA differ in M. florum compared to other bacteria?

The genomic organization around the purA gene in M. florum reflects the general trend of genome compaction observed in this organism. Given M. florum's genomic simplicity, genes involved in related metabolic pathways are often organized in transcriptional units (TUs), with common promoter motifs.

Based on transcriptomic analysis of M. florum, genes with related functions typically show coordinated expression patterns, suggesting their organization into functional operons . The transcriptome data reveals that genes included in TUs generally display significantly higher expression values compared to orphan coding sequences . While specific information about purA's position within the M. florum genome is not provided in the search results, the gene would likely be found in a TU with other purine metabolism genes for efficient transcriptional regulation.

What expression systems are most compatible for recombinant M. florum purA production?

Successful heterologous expression of M. florum purA requires consideration of several factors given the unique characteristics of this near-minimal bacterium. Based on available genetic engineering tools for M. florum, several expression approaches can be recommended:

For homologous expression within M. florum itself:

• The oriC-based plasmids developed specifically for M. florum provide an excellent foundation for expression .

• Plasmids harboring both the rpmH-dnaA and dnaA-dnaN intergenic regions have shown stable maintenance and transformation frequencies of ~4.1 × 10^-6 transformants per viable cell .

• Demonstrated resistance markers include tetracycline, puromycin, and spectinomycin/streptomycin, which can be used for selection .

For heterologous expression in E. coli or other hosts:

• Codon optimization may be necessary due to M. florum's AT-rich genome.

• Expression should account for M. florum's optimal growth temperature of 34°C, which may affect protein folding when expressed in other systems .

How does purA activity correlate with M. florum's rapid growth rate?

M. florum demonstrates an impressive doubling time of approximately 30.8 ± 2.9 minutes as measured by flow cytometry and 32.7 ± 0.9 minutes as determined by colony-forming units . This rapid growth rate necessitates efficient nucleotide biosynthesis, where purA plays a central role.

The relationship between purA activity and growth rate in M. florum likely follows patterns observed in other fast-growing bacteria, where purine biosynthetic enzymes must operate with high efficiency to support rapid DNA and RNA synthesis. Proteomic analysis of M. florum revealed that central carbon metabolism and membrane transport categories display particularly important proteome fractions (7.5% and 7.4% of protein diversity, respectively) , which would support the rapid energy production and substrate uptake required for nucleotide synthesis.

Researchers investigating this relationship should consider:

• Measuring purA expression levels throughout different growth phases

• Correlating enzyme activity with growth rate under various nutrient conditions

• Examining rate-limiting steps in the purine biosynthesis pathway in this minimal system

What structural features distinguish M. florum purA from homologs in other bacterial species?

While specific structural data for M. florum purA is not provided in the search results, several characteristics can be inferred based on its minimal genomic context and evolutionary position.

The adenylosuccinate synthetase from M. florum would likely retain the core catalytic domains found in all purA enzymes while potentially showing adaptations that reflect:

• Adaptation to M. florum's fast growth rate

• Possible optimizations for efficiency in a minimal cellular environment

• Sequence adaptations consistent with M. florum's AT-rich genome

Researchers should investigate:

• Whether M. florum purA exhibits higher catalytic efficiency than homologs

• If substrate binding sites show modifications that reflect the specific metabolite concentrations in M. florum

• Potential structural simplifications that align with the minimal nature of this organism

What are the predicted kinetic parameters of M. florum purA compared to other bacterial homologs?

Based on comparative analysis of adenylosuccinate synthetases from various bacterial species, the following kinetic parameters would be expected for M. florum purA:

The temperature optimum would likely align with M. florum's optimal growth temperature of 34°C . The relatively fast doubling time of M. florum (approximately 31 minutes) suggests that its metabolic enzymes, including purA, may have evolved for higher efficiency compared to homologs from slower-growing bacteria.

Expression Protocol:

For heterologous expression in E. coli:

• Clone the M. florum purA gene into a pET vector system with a His-tag for purification

• Transform into BL21(DE3) E. coli strain

• Grow cultures at 37°C to OD600 of 0.6-0.8

• Induce with 0.5 mM IPTG

• Shift temperature to 30°C (closer to M. florum's optimal growth temperature of 34°C)

• Continue expression for 4-6 hours or overnight

For homologous expression in M. florum:

• Clone purA into one of the developed oriC-based plasmids containing both rpmH-dnaA and dnaA-dnaN intergenic regions

• Include an appropriate selection marker (tetracycline, puromycin, or spectinomycin/streptomycin)

• Transform using polyethylene glycol-mediated transformation (frequency ~4.1 × 10^-6 transformants per viable cell) or electroporation (frequency up to 7.87 × 10^-6)

• Grow cultures at 34°C in appropriate medium

Purification Protocol:

• Harvest cells and resuspend in buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol)

• Lyse cells via sonication or French press

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Purify using Ni-NTA affinity chromatography for His-tagged protein

• Further purify by size exclusion chromatography if needed

• Assess purity by SDS-PAGE (expected molecular weight ~47-50 kDa)

• Confirm activity using the standard coupled spectrophotometric assay

What assay methods are most effective for measuring M. florum purA activity?

Three complementary methods are recommended for comprehensive characterization of M. florum purA activity:

Method 1: Spectrophotometric Coupled Assay

This standard method measures AMP production by coupling it to additional enzymes:

• Reaction mixture (1 mL): 50 mM HEPES (pH 7.5), 150 mM KCl, 10 mM MgCl2, 0.2 mM IMP, 1 mM GTP, 5 mM aspartate, 0.2 mM NADH, 2 units myokinase, 2 units pyruvate kinase, 2 units lactate dehydrogenase

• Initiate reaction by adding purified purA enzyme

• Monitor decrease in absorbance at 340 nm as NADH is oxidized

• Calculate activity based on the rate of NADH consumption (ε340 = 6,220 M^-1 cm^-1)

Method 2: HPLC Analysis of Reaction Products

For direct measurement of adenylosuccinate formation:

• Reaction mixture (100 μL): 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM IMP, 1 mM GTP, 5 mM aspartate

• Incubate with purA enzyme at 34°C (M. florum's optimal growth temperature)

• Terminate reaction at various timepoints by adding 10 μL 2.5 M HClO4

• Neutralize with K2CO3 and remove precipitate

• Analyze by HPLC with UV detection at 254 nm

• Quantify adenylosuccinate formation using standard curves

Method 3: Isothermal Titration Calorimetry (ITC)

For detailed thermodynamic analysis of substrate binding:

• Prepare purA enzyme (20-50 μM) in buffer (50 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2)

• Titrate with substrate solutions (IMP, GTP, or aspartate)

• Perform experiments at 34°C to match M. florum's physiological temperature

• Extract binding constants (Kd) and thermodynamic parameters

How can site-directed mutagenesis be used to investigate catalytic mechanisms of M. florum purA?

Site-directed mutagenesis represents a powerful approach to probe structure-function relationships in M. florum purA. Based on conserved residues in adenylosuccinate synthetases, the following experimental design is recommended:

Key Residues for Mutagenesis:

• Catalytic loop residues: Typically contain conserved aspartate and threonine residues essential for Mg2+ coordination

• IMP binding site: Often contains conserved glycine-rich regions

• GTP binding site: Look for conserved basic residues (lysine, arginine)

• Aspartate binding site: Often involves positively charged residues

Mutagenesis Protocol:

• Use a plasmid containing M. florum purA as template

• Design primers incorporating desired mutations

• Perform PCR using a high-fidelity polymerase

• Digest template DNA with DpnI

• Transform into competent E. coli

• Sequence verify mutations

• Express and purify mutant proteins

For expression in M. florum itself, utilize the developed oriC-based plasmids with appropriate mutation sites.

Analysis of Mutants:

Create a comprehensive analysis comparing wild-type and mutant enzymes:

How can recombinant M. florum purA contribute to synthetic biology efforts?

M. florum is particularly valuable for synthetic biology due to its near-minimal genome (~800 kb), fast growth rate, and lack of pathogenic potential . Understanding and manipulating purA in this context offers several opportunities:

• Minimal Cell Design: As a component of a critical metabolic pathway, purA characterization helps define the minimal gene set required for cellular function. This aligns with efforts to build simplified cellular chassis for synthetic biology .

• Metabolic Engineering: Manipulating purA expression or activity could optimize nucleotide metabolism in synthetic minimal cells, potentially improving growth rates or cellular productivity.

• Orthogonal Metabolism: Engineered variants of M. florum purA could be designed to utilize non-natural substrates, creating orthogonal metabolic pathways for synthetic biology applications.

• Biosensor Development: purA-based reporters could serve as sensors for metabolic state, detecting changes in purine nucleotide pools or related metabolites.

Researchers can leverage the developed genetic tools for M. florum, including oriC-based plasmids and transformation methods (PEG-mediated, electroporation, and conjugation) , to implement these applications.

What considerations are important when comparing M. florum purA activity to homologs from other bacterial species?

When conducting comparative enzymatic studies of purA across bacterial species, researchers should consider:

• Evolutionary Context: M. florum belongs to the Mollicutes class, which has undergone extensive genome reduction. This evolutionary history may have influenced purA function compared to enzymes from bacteria with larger genomes.

• Cellular Environment: The intracellular environment of M. florum differs from other bacteria in terms of:

  • Metabolite concentrations

  • Macromolecular crowding effects

  • Cofactor availability

  • pH and ionic strength

• Methodological Standardization: Ensure consistent experimental conditions:

  • Adjust assay temperatures to each organism's optimal growth temperature (34°C for M. florum)

  • Account for differences in pH optima

  • Use equivalent buffer conditions

• Structural Comparisons: Beyond kinetic parameters, examine:

  • Thermal stability profiles

  • Substrate specificity

  • Allosteric regulation mechanisms

  • Oligomeric state

• Genomic Context: Consider how differences in genomic architecture might influence enzyme expression and regulation. M. florum's transcriptome analysis indicates specific promoter motifs and organization of transcription units that may differ from other bacteria .

How might the minimal genome context of M. florum influence purA function and regulation?

The near-minimal nature of M. florum's genome (~800 kb) likely has profound effects on purA function and regulation:

What are the most common issues when working with recombinant M. florum purA and how can they be addressed?

Researchers working with recombinant M. florum purA may encounter several challenges:

Challenge 1: Low Expression Yields

Potential solutions:

• Optimize codon usage for the expression host

• Lower induction temperature to 30°C to improve protein folding

• Consider using a solubility-enhancing tag (e.g., MBP or SUMO)

• Test expression in M. florum using the developed oriC-based plasmids if other systems fail

Challenge 2: Protein Instability

Potential solutions:

• Include glycerol (10-20%) in all buffers

• Add reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Maintain temperature at or below 4°C during purification

• Consider adding nucleotides (IMP, GTP at 0.1-0.5 mM) to stabilize the enzyme

Challenge 3: Inconsistent Activity Measurements

Potential solutions:

• Ensure Mg2+ concentration is optimal (typically 5-10 mM)

• Verify pH is appropriate (test range 7.0-7.5)

• Check for inhibitory compounds in buffers

• Perform reactions at 34°C to match M. florum's optimal growth temperature

• Ensure all coupling enzymes are in excess when using coupled assays

Challenge 4: Difficulties with Transformation into M. florum

Potential solutions:

• Use oriC-based plasmids containing both rpmH-dnaA and dnaA-dnaN intergenic regions

• Try alternative transformation methods: PEG-mediated transformation, electroporation (up to 7.87 × 10^-6 transformants per viable cell), or conjugation from E. coli (up to 8.44 × 10^-7 transformants per viable cell)

• Verify selection using appropriate antibiotics (tetracycline, puromycin, or spectinomycin/streptomycin)

How can researchers validate that recombinant M. florum purA maintains native structural and functional properties?

To ensure that recombinant purA accurately represents the native M. florum enzyme, researchers should perform the following validation steps:

• Circular Dichroism (CD) Spectroscopy

  • Compare secondary structure elements to predicted models

  • Perform thermal denaturation studies to determine Tm value

  • Expected Tm should align with M. florum's optimal growth temperature (34°C)

• Size Exclusion Chromatography

  • Verify oligomeric state (typically dimeric for adenylosuccinate synthetases)

  • Check for aggregation or abnormal elution profiles

• Kinetic Parameter Comparison

  • If possible, compare recombinant enzyme to native purA extracted from M. florum

  • Verify that kinetic parameters fall within expected ranges

  • Ensure substrate specificity matches predicted patterns

• Mass Spectrometry

  • Confirm the exact mass of the purified protein

  • Check for post-translational modifications

  • Verify intact N- and C-termini

• In vivo Complementation

  • Test if recombinant purA can complement a purA-deficient strain

  • Measure growth rates in minimal media requiring de novo purine synthesis

• Substrate Specificity Profiling

  • Test activity with various substrate analogs

  • Compare specificity pattern to other characterized adenylosuccinate synthetases