Recombinant Acholeplasma laidlawii Glucose-6-phosphate isomerase (pgi)

What is Acholeplasma laidlawii Glucose-6-phosphate isomerase and what is its biological function?

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9), also known as phosphoglucose isomerase (PGI) or phosphohexose isomerase (PHI), is a ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells . In Acholeplasma laidlawii (strain PG-8A), this enzyme plays a crucial role in central carbon metabolism, enabling the organism to utilize glucose as an energy source.

The reaction catalyzed can be represented as:
$\text{D-glucopyranose-6-phosphate} \rightleftarrows \text{D-fructofuranose-6-phosphate}$

The enzyme participates in both glycolysis (glucose breakdown) and gluconeogenesis (glucose synthesis), making it essential for cellular energy metabolism .

What are the recommended storage and handling conditions for recombinant A. laidlawii PGI?

For optimal stability and activity retention of recombinant A. laidlawii Glucose-6-phosphate isomerase, follow these evidence-based storage and handling guidelines:

• Storage temperature: Store at -20°C; for extended storage, conserve at -20°C or -80°C .

• Reconstitution protocol:

  • Centrifuge the vial briefly before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration for long-term storage (50% is commonly used)

  • Prepare aliquots to avoid repeated freeze-thaw cycles

• Working solution stability: Working aliquots can be stored at 4°C for up to one week .

• Freeze-thaw considerations: Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of enzymatic activity .

• Shelf life expectations:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

How can I express and purify recombinant A. laidlawii PGI for research applications?

Based on established protocols for similar phosphoglucose isomerases, the following methodological approach is recommended for expression and purification of recombinant A. laidlawii PGI:

Expression system options:

• Baculovirus expression system: The commercial recombinant A. laidlawii PGI is produced using a baculovirus expression system, which often yields properly folded, active eukaryotic proteins with post-translational modifications .

• E. coli expression alternative: Similar to the approach used for M. tuberculosis PGI, the gene encoding A. laidlawii PGI can be PCR amplified and cloned into an expression vector (e.g., pET-22b(+)) under the control of an inducible promoter like T7. Expression in E. coli can be induced with IPTG, though the protein may partially form inclusion bodies .

Purification protocol:

• Harvest cells and lyse using appropriate buffer systems

• Separate soluble fraction from inclusion bodies by centrifugation

• For the soluble fraction, employ ion-exchange chromatography as a primary purification step

• Consider adding affinity chromatography if a His-tag or other fusion tag is incorporated

• Assess purity using SDS-PAGE (target >85% purity)

• Confirm identity and activity through enzymatic assays and/or mass spectrometry

For inclusion bodies, additional refolding steps would be necessary to recover active enzyme, typically involving solubilization with denaturants followed by controlled refolding through dialysis.

What assay methods can be used to measure the enzymatic activity of recombinant A. laidlawii PGI?

Several established methodological approaches can be employed to measure the enzymatic activity of recombinant A. laidlawii PGI:

1. Coupled enzyme assay (standard method):
This spectrophotometric method measures the conversion of fructose-6-phosphate (F6P) to glucose-6-phosphate (G6P), which is further converted by glucose-6-phosphate dehydrogenase (G6PDH) to 6-phosphogluconate with concurrent reduction of NAD(P)+ to NAD(P)H. The increase in absorbance at 340 nm due to NAD(P)H formation is proportional to PGI activity.

Reaction scheme:
$\text{F6P} \xrightarrow{\text{PGI}} \text{G6P} \xrightarrow{\text{G6PDH}} \text{6-phosphogluconate} + \text{NAD(P)H}$

Protocol parameters:

• Buffer: Typically Tris-HCl (pH 7.5-9.0)

• Substrate concentration: 0.1-1.0 mM F6P

• Coupling enzyme: Excess G6PDH to ensure non-rate-limiting

• Cofactor: NAD+ or NADP+ (typically 0.5-1.0 mM)

• Temperature: 25-37°C

2. Fluorescence-based high-throughput assay:
For screening inhibitors or performing kinetic studies, a fluorescence-based assay can be developed by coupling the activities of PGI with G6PDH and diaphorase, as demonstrated for Leishmania mexicana PGI :

$\text{F6P} \xrightarrow{\text{PGI}} \text{G6P} \xrightarrow{\text{G6PDH}} \text{6-phosphogluconate} + \text{NADPH} \xrightarrow{\text{Diaphorase}} \text{NAD}^+ + \text{Fluorescent product}$

3. Direct determination of substrates/products:
For more detailed kinetic analysis, direct determination of F6P or G6P can be performed using HPLC or enzymatic end-point methods.

How does the kinetic profile of A. laidlawii PGI compare with PGI from other organisms?

While specific kinetic parameters for A. laidlawii PGI are not provided in the available search results, comparisons can be made based on data from related PGIs:

Comparative Kinetic Parameters of PGIs from Different Species:

For comprehensive characterization of A. laidlawii PGI, researchers should determine:

• Km and Vmax for both forward and reverse reactions

• pH optimum and pH stability profile

• Temperature optimum and thermal stability

• Effects of potential inhibitors, particularly 6-phosphogluconate, which is known to inhibit PGIs from other species

• Influence of various ions on enzymatic activity

These parameters would help establish the specific adaptations of A. laidlawii PGI and its evolutionary relationship to PGIs from other organisms.

What is the evolutionary significance of A. laidlawii PGI in the context of Mollicutes biology?

A. laidlawii belongs to the Mollicutes class, which includes organisms with reduced genomes that have undergone degenerative evolution. Several features make A. laidlawii PGI and its metabolic context evolutionarily significant:

• Genomic context: A. laidlawii possesses the longest genome (1,496,992-bp circular chromosome) among Mollicutes with known nucleotide sequences, suggesting it may retain more ancestral metabolic capabilities than other Mollicutes .

• Genetic code usage: A. laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code (UGA as stop codon rather than coding for tryptophan), representing a more ancestral state compared to other Mollicutes .

• Metabolic capabilities: Unlike most Mollicutes, A. laidlawii has retained the capacity to synthesize saturated fatty acids de novo, suggesting its central carbon metabolism, including the role of PGI, may be more complete than in other Mollicutes .

• Evolutionary relationships: It has been suggested that acholeplasmas are evolutionary ancestors of phytoplasmas that have evolved by further degenerative evolution, making A. laidlawii PGI potentially more similar to ancestral forms than PGIs from other Mollicutes .

• Regulatory potential: A. laidlawii retains genes for polymerase type I, SOS response, signal transduction systems, RNA regulatory elements, riboswitches, and T boxes, demonstrating significant capability for regulation of gene expression and mutagenic response to stress .

The study of A. laidlawii PGI thus provides insights into the minimal functional requirements for glycolytic pathways and how essential metabolic enzymes adapt during reductive genome evolution.

How can recombinant A. laidlawii PGI be used to investigate host-pathogen interactions?

Recombinant A. laidlawii PGI offers several research avenues for investigating host-pathogen interactions:

• Immunological studies: As a conserved metabolic enzyme with potential surface exposure, PGI may elicit host immune responses. Recombinant A. laidlawii PGI can be used to:

  • Assess antibody responses in infected hosts

  • Evaluate T-cell responses to PGI epitopes

  • Determine cross-reactivity with host PGI, potentially revealing autoimmune mechanisms

• Phytopathogenic effects: A. laidlawii has been reported to cause phytopathogenic effects similar to phytoplasma infections . Recombinant PGI could be used to:

  • Investigate whether external application of PGI affects plant cell metabolism

  • Study if PGI contributes to symptom development in plants

  • Develop diagnostic tools for A. laidlawii infection in plants

• Metabolic manipulation: Understanding how A. laidlawii PGI functions in the context of host cell metabolism could reveal:

  • Competition for metabolic intermediates between host and pathogen

  • Alterations in host glycolytic flux during infection

  • Potential metabolic bottlenecks that could be targeted therapeutically

• Structural biology approaches: Comparing the structure of A. laidlawii PGI with host PGI could:

  • Identify unique structural features that could be targeted by selective inhibitors

  • Reveal evolutionary adaptations specific to the A. laidlawii lifestyle

  • Guide rational design of diagnostic tools or therapeutic interventions

What strategies can be employed to develop inhibitors specific to microbial PGIs while sparing human PGI?

The development of selective inhibitors targeting microbial PGIs while sparing human PGI represents a challenging but potentially fruitful research direction:

• Structural-based design approaches:

  • Computational analysis of cavities present on PGI's crystallographic structure can suggest potential binding sites for mixed-type inhibition mechanisms

  • Molecular docking studies comparing A. laidlawii PGI with human PGI can identify unique binding pockets

  • Fragment-based drug design focusing on regions of structural divergence

• High-throughput screening methodologies:

  • Develop fluorescence-based coupled enzyme assays similar to those used for Leishmania mexicana PGI

  • Screen compound libraries against both microbial and human PGIs in parallel to identify selective hits

  • Employ counterscreens to eliminate compounds with general protein-binding properties

• Rational design considerations:

  • Focus on nonphosphorylated inhibitors, which may offer advantages in terms of pharmacokinetics and cellular penetration compared to phosphorylated compounds

  • Target allosteric sites rather than the highly conserved active site

  • Explore the possibility of covalent inhibitors that exploit unique cysteine residues present in microbial but not human PGIs

• Selectivity challenges:

  • Current inhibitors of trypanosomatid PGIs also inhibit human PGI , highlighting the need for more selective approaches

  • The conserved nature of the PGI active site across species presents a significant challenge

  • Kinetic differences between microbial and human PGIs (optimal pH, temperature, etc.) could be exploited

What factors might affect the activity and stability of recombinant A. laidlawii PGI in experimental settings?

Several factors can influence the activity and stability of recombinant A. laidlawii PGI in laboratory experiments:

• Buffer composition effects:

  • pH sensitivity: While optimal pH for A. laidlawii PGI is not specified in the search results, other PGIs (like M. tuberculosis PGI) show optimal activity at pH 9.0

  • Ionic strength: High salt concentrations may affect protein solubility and activity

  • Buffer components: Some buffers contain compounds that may inhibit enzyme activity

• Temperature considerations:

  • Thermal stability: Storage at -20°C or -80°C is recommended; working aliquots at 4°C

  • Reaction temperature: While optimal temperature is not specified for A. laidlawii PGI, related enzymes function optimally at 37°C

  • Freeze-thaw cycles: Repeated freezing and thawing should be avoided

• Protein modification issues:

  • Oxidation of cysteine residues can affect activity

  • Proteolytic degradation during storage or assay

  • Potential post-translational modifications affecting function (A. laidlawii proteins can undergo phosphorylation and acylation)

• Cofactor and substrate considerations:

  • Substrate purity: Commercial preparations of F6P or G6P may contain inhibitory contaminants

  • Metal ion effects: While some PGIs don't require metal ions for activity , trace metal contamination could influence results

  • Competitive inhibitors: 6-phosphogluconate is a known inhibitor of many PGIs

How can genetic manipulation techniques be applied to study A. laidlawii PGI function in vivo?

Several genetic approaches can be employed to investigate A. laidlawii PGI function in its native context:

• Transformation strategies:

  • Polyethylene glycol (PEG)-induced transformation has been demonstrated in A. laidlawii, enabling introduction of genetic material

  • Protoplast fusion, which showed higher recombination frequency than transformation in A. laidlawii, can be used to introduce genetic modifications

• Gene knockout/knockdown approaches:

  • As PGI is likely essential for glycolysis, conditional knockout systems might be necessary

  • Antisense RNA or CRISPR interference could provide tunable reduction in PGI expression

  • Temperature-sensitive mutants could be generated to study PGI function under restrictive conditions

• Site-directed mutagenesis applications:

  • Specific residues in the active site can be mutated to study structure-function relationships

  • Introduction of reporter tags (like fluorescent proteins) to study localization

  • Creation of catalytically inactive variants to study potential non-enzymatic functions

• Reporter systems development:

  • Fusion of promoter regions to reporter genes to study transcriptional regulation

  • Construction of translational fusions to study protein expression levels

  • Integration of biosensors to monitor metabolic changes related to PGI activity

• Considerations specific to A. laidlawii:

  • A REP- phenotype variant of A. laidlawii has been isolated that shows increased UV sensitivity but no change in growth kinetics , demonstrating the feasibility of generating stable variants

  • The universal genetic code usage in A. laidlawii allows direct application of genetic tools developed for other bacteria without codon optimization

What analytical techniques can be applied to characterize post-translational modifications of A. laidlawii PGI?

Proteomics studies have revealed that A. laidlawii proteins undergo post-translational modifications including phosphorylation and acylation . To characterize these modifications in recombinant or native PGI:

In A. laidlawii, 74 candidate phosphorylated proteins have been detected, and among 20 acylated proteins, 14 contained palmitic chains, and 6 contained stearic chains, with no residues of linoleic or oleic acid observed . Determining whether PGI is among these modified proteins and understanding the functional consequences of these modifications would provide valuable insights into metabolic regulation in this organism.

How does A. laidlawii PGI compare structurally and functionally with PGI enzymes from other bacterial species?

A comparative analysis of PGI enzymes from different bacterial species reveals important structural and functional variations:

• Primary structure comparisons:

  • A. laidlawii PGI consists of 423 amino acids

  • Interspecies variation at the primary structure level is common among PGIs and can produce heterogeneity in structure and function

  • Sequence alignment analysis would reveal conserved catalytic residues versus species-specific regions

• Functional parameters:

  • M. tuberculosis PGI exhibits optimal activity at 37°C and pH 9.0

  • M. tuberculosis PGI has a specific activity of 600 U/mg protein and Km of 0.318 mM for fructose-6-phosphate

  • M. tuberculosis PGI does not require mono- or divalent cations for activity

  • Comparative kinetic studies with A. laidlawii PGI would reveal adaptations specific to the Acholeplasma lifestyle

• Structural features:

  • Most bacterial PGIs are homodimers, though specific quaternary structure of A. laidlawii PGI is not described in the search results

  • The catalytic mechanism is generally conserved across species, involving acid-base catalysis

  • Species-specific structural adaptations may relate to environmental conditions and metabolic demands

• Inhibition profiles:

  • 6-phosphogluconate inhibits M. tuberculosis PGI with a Ki of 0.8 mM

  • Inhibition studies with A. laidlawii PGI would provide insights into regulatory mechanisms and potential differences in substrate binding pocket

What insights can be gained from studying A. laidlawii PGI in the context of bacterial evolution and adaptation?

Studying A. laidlawii PGI offers unique perspectives on bacterial evolution and adaptation:

• Genomic streamlining context:

  • A. laidlawii has undergone genomic streamlining as part of the Mollicutes evolution yet retains a relatively large genome (1,496,992 bp) compared to other Mollicutes

  • Retention of PGI functionality despite genome reduction highlights its essential metabolic role

  • Comparison with more reduced Mollicutes genomes can reveal the minimal requirements for glycolysis

• Evolutionary trajectory insights:

  • A. laidlawii is suggested to be an evolutionary ancestor of phytoplasmas

  • Comparing A. laidlawii PGI with phytoplasma PGIs could reveal evolutionary patterns in enzyme modification

  • The use of universal genetic code in A. laidlawii (unlike most Mollicutes) positions its proteins, including PGI, at an interesting evolutionary juncture

• Metabolic adaptation evidence:

  • A. laidlawii's ability to synthesize fatty acids de novo (unlike most Mollicutes) suggests its central carbon metabolism, including PGI function, may be more versatile

  • Understanding how PGI function is preserved despite genome reduction provides insights into metabolic priorities during bacterial adaptation

  • Potential moonlighting functions of PGI beyond glycolysis could reveal additional selective pressures

• Horizontal gene transfer considerations:

  • A. laidlawii can undergo transformation and fusion of protoplasts , potentially facilitating horizontal gene transfer

  • Analysis of PGI sequence could reveal evidence of recombination events

  • Comparison with PGIs from other bacteria might identify potential horizontal gene transfer events in evolutionary history

How might differences between A. laidlawii PGI and human PGI be exploited for the development of selective antimicrobial strategies?

The development of selective antimicrobial strategies targeting A. laidlawii PGI while sparing human PGI requires understanding key differences between these enzymes:

• Structural divergence exploration:

  • Detailed structural comparison between A. laidlawii PGI and human PGI could reveal unique binding pockets

  • Computational cavity analysis, similar to that performed for Leishmania mexicana PGI , could identify potential binding sites unique to bacterial PGIs

  • X-ray crystallography or cryo-EM studies of A. laidlawii PGI would provide foundation for structure-based drug design

• Kinetic and mechanistic differences:

  • Differences in substrate affinity, catalytic efficiency, or allosteric regulation could be exploited

  • pH optima differences (bacterial PGIs often function at higher pH than human PGI)

  • Temperature stability differences reflecting the different physiological environments

• Inhibitor development strategies:

  • Nonphosphorylated inhibitors may offer advantages in terms of pharmacokinetics

  • Mixed-type inhibition mechanisms targeting both the active site and unique allosteric sites

  • Covalent inhibitors exploiting unique cysteine residues present only in bacterial PGIs

• Selective targeting challenges:

  • Current inhibitors of trypanosomatid PGIs also inhibit human PGI , indicating the difficulty of achieving selectivity

  • The conserved nature of the active site across species necessitates targeting less conserved regions

  • High-throughput screening coupled with rational design approaches may be necessary to identify leads with sufficient selectivity

• Delivery system considerations:

  • A. laidlawii lacks a cell wall but has a cell membrane , potentially offering unique opportunities for inhibitor delivery

  • Species-specific uptake mechanisms could be exploited to concentrate inhibitors in bacterial cells

  • Consideration of potential off-target effects on commensal bacteria with similar PGIs

How does A. laidlawii PGI function within the broader metabolic network of the organism?

Understanding A. laidlawii PGI within its metabolic context provides insights into cellular function and potential vulnerabilities:

What experimental approaches can be used to study the impact of PGI inhibition or mutation on A. laidlawii metabolism?

Several experimental approaches can elucidate the systemic effects of PGI inhibition or mutation:

• Metabolomics approaches:

  • Untargeted LC-MS/MS analysis to detect global metabolic changes

  • Targeted metabolite analysis focusing on glycolysis, pentose phosphate pathway, and TCA cycle intermediates

  • Stable isotope labeling to track carbon flux through central metabolic pathways

  • Time-course experiments to capture dynamic metabolic responses

• Genetic manipulation strategies:

  • Conditional knockdown systems to reduce PGI expression in a controlled manner

  • Point mutations in catalytic residues to create hypomorphic PGI variants

  • Overexpression studies to assess the effects of increased PGI activity

  • Complementation experiments with heterologous PGIs to identify species-specific functions

• Chemical inhibition methods:

  • Application of known PGI inhibitors at sub-lethal concentrations

  • Structure-based design of A. laidlawii PGI-specific inhibitors

  • Dose-response studies to correlate inhibition level with metabolic effects

  • Combination with other metabolic inhibitors to identify synthetic interactions

• Systems-level analysis techniques:

  • Transcriptomics to assess compensatory gene expression changes

  • Proteomics to identify changes in enzyme levels and modifications

  • Flux balance analysis to predict systemic effects of PGI inhibition

  • Metabolic control analysis to quantify PGI's influence on various pathways

How might post-translational modifications regulate A. laidlawii PGI activity in response to environmental changes?

Post-translational modifications (PTMs) provide a rapid mechanism for regulating enzyme activity in response to environmental changes:

• Phosphorylation regulatory potential:

  • 74 candidate phosphorylated proteins have been detected in A. laidlawii

  • Phosphorylation can rapidly alter enzyme activity by changing protein conformation or charge distribution

  • Key residues (serine, threonine, tyrosine) in or near the active site could modulate substrate binding or catalysis

  • Signal transduction systems in A. laidlawii could transmit environmental information to kinases/phosphatases targeting PGI

• Acylation regulatory mechanisms:

  • 20 acylated proteins have been identified in A. laidlawii (14 with palmitic chains, 6 with stearic chains)

  • Acylation can affect protein localization, stability, and protein-protein interactions

  • Could link PGI activity to membrane association or complex formation

  • Might connect fatty acid synthesis capabilities of A. laidlawii with glycolytic regulation

• Environmental response patterns:

  • Nutrient availability changes could trigger PTM-mediated regulation of PGI

  • Stress responses might involve reversible modification of metabolic enzymes

  • Temperature or pH fluctuations could be sensed and transmitted via PTM systems

  • Host environment adaptation could involve specific PTM patterns

• Experimental approaches to study PTM-mediated regulation:

  • Phosphoproteomic analysis under different growth conditions

  • Site-directed mutagenesis of potential PTM sites

  • In vitro modification assays with purified kinases or acyltransferases

  • Correlation of PTM status with enzymatic activity and metabolic flux

Understanding these PTM-mediated regulatory mechanisms would provide insights not only into A. laidlawii metabolism but also into the broader question of how minimal genome organisms maintain metabolic flexibility despite reduced genetic complexity.