Recombinant Lactobacillus plantarum ATP phosphoribosyltransferase regulatory subunit (hisZ)

What is the role of ATP phosphoribosyltransferase regulatory subunit (HisZ) in Lactobacillus plantarum?

HisZ functions as a regulatory subunit that works in conjunction with the catalytic subunit HisG to form the ATP phosphoribosyltransferase (ATPPRT) complex. In L. plantarum, HisZ plays a critical role in allosterically activating HisG, which catalyzes the first step in histidine biosynthesis - the condensation of ATP and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to form N¹-(5-phospho-β-D-ribosyl)-ATP (PRATP) and pyrophosphate .

This regulatory mechanism is particularly important as HisZ significantly enhances the catalytic efficiency (kcat) of HisG. Research has demonstrated that when HisZ binds to HisG, it forms a hetero-octameric holoenzyme that exhibits substantially higher catalytic activity compared to HisG alone .

How is the his gene cluster organized in Lactobacillus plantarum compared to other organisms?

In Lactobacillus plantarum, the his genes are arranged in a compact cluster with a relative gene order of hisGDBHAF(IE)C, which resembles the organization found in some bacteria and archaea. This particular arrangement differs from the complete enterobacterial his operon (hisGDC(NB)HAF(IE)) found in organisms like Escherichia coli .

The gene organization in L. plantarum shows a translocation of the hisC gene compared to the E. coli arrangement, where it moves from downstream of hisD to downstream of his(IE) at the end of the cluster. This compact organization with hisC next to his(IE) has been detected in only a few genomes, including those of Oceanobacillus iheyensis and L. plantarum .

Notably, in L. plantarum, the hisZ gene is positioned next to hisG, encoding ATP phosphoribosyltransferase, which indicates an evolutionary relationship between these functionally related genes .

What are the optimal expression systems for producing recombinant L. plantarum HisZ protein?

For the expression of recombinant L. plantarum HisZ, several expression systems have proven effective:

• pSIP Expression System: This inducible expression system designed specifically for lactic acid bacteria has been successfully used for heterologous protein expression in L. plantarum. The system utilizes signal peptides (such as the endogenous signal peptide 1320 from L. plantarum) and can be induced with synthetic peptide pheromone (SppIP) .

• pWCF-Based Vectors: These have been effectively used to express recombinant proteins in L. plantarum strains. For example, the pWCF-HA1 and pWCF-HA1-DCpep vectors have been used to construct recombinant L. plantarum strains expressing influenza virus antigens .

For optimal expression, the following considerations are important:

• Codon optimization according to the codon usage bias of L. plantarum

• Use of appropriate signal peptides for correct localization

• Selection of suitable induction conditions: 50 ng/mL SppIP at 37°C for 6-10 hours has been shown to yield high protein expression for some recombinant proteins in L. plantarum

What PCR conditions are recommended for amplifying the hisZ gene from L. plantarum genomic DNA?

Based on protocols used for similar gene amplifications in L. plantarum, the following PCR conditions are recommended:

• Denaturation: 98°C for 10 seconds

• Annealing: 65°C for 15 seconds (may need optimization based on primer design)

• Extension: 72°C for 10 seconds per kb of target sequence

• Cycles: 25-30

For successful amplification:

• Design primers with appropriate restriction sites (e.g., XbaI and HindIII) for subsequent cloning

• Include proper proofreading polymerase to minimize mutation risk

• Use genomic DNA extracted from L. plantarum using standard bacterial DNA isolation methods

• Consider adding DMSO (5-10%) if GC content is high

How can I verify successful expression of recombinant HisZ protein in L. plantarum?

Verification of successful expression can be accomplished through multiple complementary approaches:

• Western Blot Analysis: Using antibodies specific to HisZ or to added epitope tags (e.g., HA tag). This method has been successfully used to detect expression of recombinant proteins in L. plantarum .

• Flow Cytometry: Particularly useful for cell surface-displayed proteins, using specific antibodies followed by fluorescently-labeled secondary antibodies .

• Indirect Immunofluorescence: Using anti-HisZ antibodies or antibodies against epitope tags, followed by fluorescently-labeled secondary antibodies for visualization .

• Functional Assays: Measuring ATP phosphoribosyltransferase activity in cell lysates, which should increase with successful co-expression of functional HisZ and HisG .

• Mass Spectrometry: For definitive identification and characterization of the expressed protein .

How does temperature affect the catalytic activity of L. plantarum HisZG complex?

Temperature has significant effects on the catalytic activity of the HisZG complex:

• Optimal Temperature Range: While the exact optimum for L. plantarum HisZG hasn't been definitively established in the provided literature, research on homologous systems indicates that the complex functions optimally at physiological temperatures (30-37°C for mesophilic bacteria).

• Low Temperature Effects: Studies with related ATP phosphoribosyltransferases show that at low temperatures (around 5°C), the rate-limiting step changes. At 5°C, the burst of PRATP formation by the activated enzyme can be directly observed, which is too fast to detect at higher temperatures .

• Mass-Dependent Effects: In heavy-isotope substitution studies of related ATP phosphoribosyltransferases, the catalytic rate of HisZ-activated HisG decreases in a strictly mass-dependent fashion at low temperatures. This indicates that protein mass can modulate thermal motions relevant to catalysis under certain conditions .

• Temperature and Rate-Limiting Steps: At higher temperatures, product release often becomes rate-limiting for the activated complex, while the chemical step remains rate-limiting for HisG alone .

What kinetic parameters characterize the L. plantarum HisZG complex compared to HisG alone?

The allosteric activation of HisG by HisZ significantly alters the kinetic parameters of ATP phosphoribosyltransferase:

When HisG is allosterically activated by HisZ:

• The catalytic rate (kcat) increases substantially

• The rate-limiting step shifts from chemistry to product release

• A burst of product formation can be observed in pre-steady-state kinetics

• The enzyme shows altered responses to changes in metal ions, temperature, and solvent viscosity

What methods can be used to assay ATP phosphoribosyltransferase activity in recombinant L. plantarum systems?

Several methods can be employed to assay ATP phosphoribosyltransferase activity:

• Spectrophotometric Coupled Assay: Monitoring the consumption of ATP or production of pyrophosphate by coupling to enzymatic reactions that produce detectable spectroscopic changes.

• HPLC-Based Methods: Directly measuring the formation of N¹-(5-phospho-β-D-ribosyl)-ATP (PRATP) using high-performance liquid chromatography.

• Radiochemical Assays: Using radiolabeled substrates (typically ¹⁴C-ATP or ³²P-PRPP) to track product formation.

• Pre-Steady-State Kinetics: Using rapid mixing techniques (stopped-flow or quenched-flow) to measure the initial burst of product formation, which is particularly important for the activated HisZG complex .

• pH-Rate Profiling: Determining the pH dependence of enzymatic activity, which for related ATP phosphoribosyltransferases shows maximum catalysis above pH 8.0 .

To accurately determine kinetic parameters, it's important to control reaction conditions (pH, temperature, metal ion concentration) and to validate results using multiple approaches.

What structural changes occur when HisZ binds to HisG in L. plantarum?

When HisZ binds to HisG in L. plantarum and related organisms, significant structural changes occur:

• Formation of Hetero-Octameric Complex: The binding results in a hetero-octameric holoenzyme, typically comprising four HisG and four HisZ subunits .

• Allosteric Conformational Changes: The binding of HisZ to HisG induces conformational changes in the catalytic site of HisG, enhancing its catalytic efficiency.

• Altered Active Site Dynamics: The allosteric activation modifies the dynamics of the active site, affecting substrate binding, transition state stabilization, and product release.

• Modified Thermal Motions: The complex exhibits altered thermal motions compared to HisG alone, which can be detected through temperature-dependent studies and isotope-labeling experiments .

• Stabilization of Active Conformation: HisZ binding stabilizes a more catalytically competent conformation of HisG, contributing to the increased catalytic rate.

These structural changes are crucial for the allosteric regulation of ATP phosphoribosyltransferase activity, which is a key control point in histidine biosynthesis.

How do mutations in the HisZ subunit affect the activity of the HisZG complex?

Mutations in the HisZ subunit can significantly impact the activity of the HisZG complex through various mechanisms:

• Interface Disruption: Mutations at the HisZ-HisG interface can weaken binding, reducing or eliminating the allosteric activation.

• Conformational Rigidity: Mutations that increase the rigidity of HisZ can impair its ability to induce the necessary conformational changes in HisG.

• Allosteric Signal Transduction: Mutations in residues involved in transmitting the allosteric signal from HisZ to HisG can reduce activation efficiency without affecting binding.

• Thermal Motion Modulation: As protein mass modulation affects catalytic rates at low temperatures, mutations that alter local or global dynamics of HisZ can impact enzymatic function .

• Product Release Pathway: Since HisZ activation shifts the rate-limiting step to product release, mutations affecting the product exit channel can have pronounced effects on catalysis.

Research on related ATP phosphoribosyltransferases has shown that disruption of specific enzyme-product interactions can abolish isotope effects, highlighting the importance of these interactions in the catalytic cycle .

How can recombinant L. plantarum expressing HisZ be used as a research tool?

Recombinant L. plantarum expressing HisZ can serve as a valuable research tool in several contexts:

• Studying Allosteric Regulation: As a model system for investigating allosteric regulation mechanisms in enzymes, particularly how protein-protein interactions modulate catalytic activity.

• Enzyme Evolution Research: For exploring the evolutionary relationship between amino acid biosynthesis and protein synthesis pathways, as HisZ shares domain similarity with proteins involved in translation .

• Metabolic Engineering: As part of efforts to optimize histidine biosynthesis pathways for biotechnological applications.

• Structural Biology: For crystallographic and biophysical studies of protein complexes and allosteric interactions.

• Molecular Dynamics Simulations: As a target for computational studies of protein dynamics and allostery, especially with the availability of structural data .

• Protein-Protein Interaction Studies: As a model system for investigating how regulatory subunits modify catalytic subunit function through direct physical interactions.

What advantages does L. plantarum offer as a host for expressing recombinant HisZ compared to other expression systems?

Lactobacillus plantarum offers several distinct advantages as a host for expressing recombinant HisZ:

• Native Environment: As HisZ naturally occurs in L. plantarum, the organism provides the appropriate cellular environment, post-translational modifications, and potential interaction partners for proper folding and function.

• Food-Grade Safety: L. plantarum is recognized as generally safe (GRAS), making it suitable for applications where safety is a concern .

• Probiotic Potential: L. plantarum has inherent probiotic properties, which can be beneficial for certain applications combining probiotic benefits with recombinant protein expression .

• Mucosal Delivery Capabilities: L. plantarum can effectively deliver proteins to mucosal surfaces, making it valuable for immunological studies .

• Persistent Colonization: L. plantarum can colonize various host environments, allowing for sustained protein delivery in experimental settings .

• Validated Expression Systems: Well-established expression systems like pSIP and pWCF vectors are available specifically for L. plantarum .

• Modulation of Host Microbiota: L. plantarum can beneficially influence the host microbiome, which may be advantageous for certain research applications .

How does protein mass modulation of HisZ affect the allosteric regulation of ATP phosphoribosyltransferase?

Protein mass modulation of HisZ has significant implications for allosteric regulation of ATP phosphoribosyltransferase, revealing fundamental aspects of enzyme dynamics:

• Mass-Dependent Catalytic Effects: Research on related ATP phosphoribosyltransferases has shown that isotope-labeling (increasing the mass) of the catalytic subunit results in decreased catalytic rates at low temperatures when activated by the regulatory subunit. This effect is strictly mass-dependent across different subunit masses .

• Product Release Dynamics: Surprisingly, the mass effect is not linked to the chemical step but to fast motions governing product release in the activated enzyme. This indicates that protein mass can affect thermal motions relevant to catalysis beyond the bond-breaking/forming steps .

• Temperature Dependence: The mass effect is most pronounced at low temperatures, suggesting a complex interplay between thermal energy, protein dynamics, and catalytic function .

• Allosteric Communication: The mass-dependent effects reveal how allosteric signals propagate through the protein complex, affecting dynamics at sites distant from the protein-protein interface.

• Specific Interactions: Disruption of specific enzyme-product interactions has been shown to abolish isotope effects, highlighting the importance of these interactions in the catalytic cycle .

These findings suggest that allosteric regulation involves not just static conformational changes but also modulation of protein dynamics across multiple timescales.

What are the evolutionary implications of the relationship between HisZ and proteins involved in translation?

The evolutionary relationship between HisZ and proteins involved in translation has profound implications:

• Common Ancestry: HisZ shares domain similarity with proteins involved in translation, suggesting a common evolutionary origin for amino acid biosynthesis and protein synthesis pathways .

• Ancient Metabolic Connection: This relationship implies an early connection between the biosynthesis of amino acids and proteins, potentially dating back to the early evolution of cellular life.

• Functional Repurposing: The adaptation of translation-related protein domains for metabolic regulation represents a case of evolutionary repurposing of protein functions.

• Regulatory Network Evolution: The connection suggests that regulatory networks controlling amino acid synthesis and protein synthesis may have evolved in concert.

• Metabolic-Translational Coupling: The relationship between HisZ and translation proteins may reflect ancient coupling mechanisms between amino acid availability and protein synthesis rates.

• Domain Conservation: Despite functional divergence, structural conservation between HisZ and certain translation factors points to constraints on protein architecture that persist through evolutionary time.

This evolutionary connection provides insight into the fundamental relationships between central cellular processes and may inform our understanding of the earliest stages of cellular evolution.

What are common challenges in the heterologous expression of L. plantarum HisZ and how can they be addressed?

Heterologous expression of L. plantarum HisZ presents several challenges that researchers should anticipate:

• Codon Usage Bias:

  • Problem: Suboptimal codon usage in heterologous hosts can lead to poor expression.

  • Solution: Optimize codons according to the expression host's bias. For expression in L. plantarum itself, this may not be necessary .

• Protein Solubility:

  • Problem: Recombinant HisZ may form inclusion bodies in some expression systems.

  • Solution: Express at lower temperatures (15-30°C), use solubility-enhancing fusion tags, or optimize induction conditions (e.g., lower inducer concentration, 50 ng/mL SppIP for L. plantarum) .

• Protein-Protein Interactions:

  • Problem: HisZ function depends on interaction with HisG, which may be absent in heterologous hosts.

  • Solution: Co-express HisG and HisZ from L. plantarum together for functional studies .

• Protein Verification:

  • Problem: Confirming expression and functionality can be challenging.

  • Solution: Use multiple detection methods, including Western blotting, functional assays, and mass spectrometry .

• Stability Issues:

  • Problem: Recombinant proteins may be unstable in some conditions.

  • Solution: Optimize storage conditions and buffer composition; consider testing stability under different pH, temperature, and salt concentration conditions .

How can I optimize the co-expression of HisZ and HisG to obtain a functional recombinant HisZG complex?

Optimizing co-expression of HisZ and HisG requires careful consideration of several factors:

• Vector Design:

  • Use compatible vectors with different selectable markers for co-expression

  • Consider using a single vector with multiple expression cassettes

  • Ensure balanced expression levels of both proteins

• Expression Timing:

  • Synchronize expression of both proteins by using the same promoter system

  • If using inducible promoters, optimize inducer concentration (e.g., 50 ng/mL SppIP has been effective for some recombinant proteins in L. plantarum)

• Stoichiometry Control:

  • Adjust relative promoter strengths if one protein tends to be overexpressed

  • Monitor expression levels of both proteins to ensure proper complex formation

• Verification of Complex Formation:

  • Use co-immunoprecipitation to confirm HisZ-HisG interaction

  • Employ size-exclusion chromatography to isolate the hetero-octameric complex

  • Verify complex formation through activity assays comparing HisG alone vs. the HisZG complex

• Purification Strategy:

  • Consider tandem affinity purification using different tags on HisZ and HisG

  • Use mild purification conditions to preserve complex integrity

  • Include glycerol (5-10%) in buffers to stabilize the complex

Successful co-expression should result in significantly enhanced ATP phosphoribosyltransferase activity compared to expression of HisG alone, which can serve as a functional verification of proper complex formation .

What analytical techniques are most suitable for studying the HisZ-HisG interaction and its effects on enzyme kinetics?

Several analytical techniques are particularly valuable for studying the HisZ-HisG interaction and its kinetic effects:

• Steady-State Enzyme Kinetics:

  • Measurement of kinetic parameters (Km, kcat, kcat/Km) under varying conditions

  • pH-rate profiling to determine optimal pH and ionization states

  • Metal ion dependence studies to assess cofactor requirements

  • Temperature dependence studies to determine activation parameters

• Pre-Steady-State Kinetics:

  • Stopped-flow spectroscopy to detect transient species and measure fast reactions

  • Quenched-flow techniques for chemical quenching at defined time points

  • Single-turnover experiments to isolate individual steps in the catalytic cycle

• Biophysical Interaction Analysis:

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Microscale thermophoresis (MST) for interaction studies in complex environments

• Structural Techniques:

  • X-ray crystallography to determine atomic-resolution structures

  • Small-angle X-ray scattering (SAXS) for solution structure analysis

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational dynamics

  • Cryo-electron microscopy for visualization of complex architectures

• Advanced Spectroscopic Methods:

  • Nuclear magnetic resonance (NMR) for studying protein dynamics

  • Fluorescence resonance energy transfer (FRET) to measure distances between labeled sites

  • Circular dichroism (CD) to monitor secondary structure changes upon complex formation

• Mass-Modulation Studies:

  • Heavy isotope labeling to probe the role of protein dynamics in catalysis

  • Temperature-dependent kinetic isotope effects to identify dynamically coupled processes