Recombinant Lactobacillus plantarum Undecaprenyl pyrophosphate synthase (uppS)

What is Undecaprenyl pyrophosphate synthetase and what is its role in bacterial cell wall synthesis?

Undecaprenyl pyrophosphate synthetase (Upp synthetase) is a prenyltransferase that catalyzes the synthesis of undecaprenyl pyrophosphate (UPP, C55-PP), a lipid carrier containing a trans,cis-mixed isoprenoid chain. UPP serves as an essential lipid carrier for glycosyl transfer in the biosynthesis of various cell wall polysaccharide components in bacteria. In the isoprenoid biosynthetic pathway, Upp synthetase uses farnesyl pyrophosphate (FPP) as the starting molecule for further rounds of sequential condensation with isopentenyl pyrophosphate (IPP) to generate UPP. This enzyme plays a crucial role in bacterial cell wall synthesis, making it essential for bacterial growth and survival.

How does L. plantarum Upp synthetase compare structurally and functionally to homologs from other bacterial species?

L. plantarum Upp synthetase shares significant structural and functional similarities with homologs from other bacterial species. Studies have shown that Upp synthetase genes and their corresponding enzymes have been identified and characterized in multiple bacterial species including Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae. While L. plantarum Upp synthetase has been studied extensively from a biochemical perspective, the functional characteristics appear consistent across species. Like other bacterial homologs, L. plantarum Upp synthetase requires detergents such as Triton X-100 and divalent metal ions such as Mg²⁺ for activity. The enzyme follows similar catalytic mechanisms across species, involving the sequential addition of IPP units to the growing isoprenoid chain.

What are the primary biochemical characteristics of recombinant L. plantarum Upp synthetase?

Recombinant L. plantarum Upp synthetase exhibits several key biochemical characteristics:

• Detergent dependency: The enzyme requires Triton X-100 for activity

• Divalent metal ion requirement: MgCl₂ is essential for catalytic function

• Substrate specificity: Uses FPP as the allylic substrate and IPP as the homoallylic substrate

• Product characteristics: Generates UPP with a trans,cis-mixed isoprenoid chain (C55-PP)

• Catalytic mechanism: Performs sequential condensation reactions to extend the isoprenoid chain

These characteristics are largely consistent with Upp synthetases from other bacterial species, suggesting a high degree of conservation in the functional properties of this enzyme class across diverse bacterial taxa.

What are the most effective strategies for cloning the uppS gene from L. plantarum?

For successful cloning of the uppS gene from L. plantarum, researchers should consider the following methodological approach:

• Genomic DNA isolation: Use specialized protocols designed for Gram-positive bacteria, which typically require more rigorous cell lysis procedures involving lysozyme and mutanolysin pretreatment.

• PCR amplification: Design primers based on the conserved regions of uppS genes from related Lactobacillus species or using the L. plantarum genome sequence. Include appropriate restriction enzyme sites at the 5' ends of primers to facilitate directional cloning.

• Vector selection: Choose an expression vector compatible with both E. coli (for cloning) and the intended expression host. Vectors containing the T7 promoter system (like pET series) or the tac promoter are typically effective.

• Sequence verification: After cloning, perform complete sequencing of the insert to confirm the absence of PCR-induced mutations.

• Codon optimization: Consider codon optimization if expressing in a heterologous host, as L. plantarum has a distinct codon usage pattern compared to common expression hosts like E. coli.

What expression systems yield the highest activity for recombinant L. plantarum Upp synthetase?

Based on research with Upp synthetases from various bacterial species, the following expression systems have proven effective for obtaining high-activity recombinant L. plantarum Upp synthetase:

For optimal activity, expression at lower temperatures (16-25°C) after induction and inclusion of 0.5-1% Triton X-100 in the lysis buffer are recommended to improve solubility and activity of the recombinant enzyme.

How does the addition of affinity tags affect the activity of recombinant L. plantarum Upp synthetase?

The addition of affinity tags, particularly His-tags, to recombinant L. plantarum Upp synthetase generally does not significantly alter the enzyme's catalytic properties. Comparative studies with His-tagged and untagged versions of Upp synthetase from E. coli found no appreciable differences in enzymatic activity or biochemical properties. Both tagged and untagged versions showed similar dependence on Triton X-100 and MgCl₂, with comparable substrate specificities and kinetic parameters.

What purification methods yield the highest purity and activity for recombinant L. plantarum Upp synthetase?

For optimal purification of recombinant L. plantarum Upp synthetase, a multi-step approach is recommended:

For His-tagged constructs:

• Cell lysis: Sonication or high-pressure homogenization in buffer containing 25 mM HEPES-OH (pH 8.0), 10% glycerol, 1 mM DTT, 1 mM PMSF, and 0.5-1% Triton X-100.

• Ni²⁺ affinity chromatography: Using Ni-NTA resin with an imidazole gradient for elution (typically 20-250 mM).

• Size exclusion chromatography: For further purification and buffer exchange using Superdex 200 or similar matrix.

For untagged constructs:

• Ammonium sulfate fractionation: Collect proteins precipitating between 35-50% saturation.

• Ion exchange chromatography: Apply to a Phospho-Ultrogel A6R column and elute with a NaCl gradient.

• Size exclusion chromatography: Final purification step using appropriate buffer (25 mM HEPES-OH, pH 8.0, with 10% glycerol, 1 mM DTT, and 0.1% Triton X-100).

Throughout all purification steps, it is essential to maintain Triton X-100 in the buffers to preserve enzyme activity. Purification yields of 15-20 mg of pure enzyme per liter of bacterial culture can typically be achieved with these methods.

What analytical methods are most suitable for assessing the activity of Upp synthetase?

Several complementary analytical methods can be employed to assess Upp synthetase activity:

• Coupled enzymatic assay: This method uses inorganic pyrophosphatase to convert the pyrophosphate released during the condensation reaction to inorganic phosphate, which can then be quantified colorimetrically using the method of Kodama et al. This approach allows for continuous monitoring of enzyme activity.

• Radiometric assay: Measures the incorporation of radiolabeled [¹⁴C]IPP or [³H]IPP into product. After reaction completion, the lipid products are extracted with n-butanol and quantified by scintillation counting. This method provides high sensitivity.

• HPLC analysis: Separates reaction products using reverse-phase HPLC with UV detection at 210 nm. This approach allows direct visualization and quantification of the polyprenyl pyrophosphate products.

• Mass spectrometry: LC-MS/MS can be used to identify and quantify reaction products with high specificity and sensitivity, particularly useful for characterizing novel variants or substrates.

• Photolabeling: Using photolabile substrate analogs like [³H]DAFTP-GDP followed by UV irradiation allows for specific labeling of the enzyme's active site, useful for structural studies.

How can researchers determine the kinetic parameters of recombinant L. plantarum Upp synthetase?

To determine the kinetic parameters of recombinant L. plantarum Upp synthetase, researchers should follow these methodological steps:

• Reaction conditions optimization:

  • Buffer: 25 mM HEPES-OH (pH 8.0)

  • Detergent: 0.1-0.5% Triton X-100

  • Divalent cation: 0.5-5 mM MgCl₂

  • Temperature: 30-37°C

  • Reducing agent: 1 mM DTT

• Initial velocity measurements:

  • For Km and Vmax determination for FPP: Vary FPP concentration (0.1-100 μM) while keeping IPP concentration fixed at saturating levels (>100 μM).

  • For Km and Vmax determination for IPP: Vary IPP concentration (0.1-100 μM) while keeping FPP concentration fixed at saturating levels (>50 μM).

  • Use the coupled assay with inorganic pyrophosphatase to measure initial velocities.

• Data analysis:

  • Plot initial velocity versus substrate concentration.

  • Fit data to appropriate enzyme kinetic models (Michaelis-Menten, Hill, or more complex models if substrate inhibition is observed).

  • Calculate Km, Vmax, and kcat using non-linear regression analysis.

  • Determine the catalytic efficiency (kcat/Km) for each substrate.

• Product analysis:

  • Analyze chain length distribution of products using HPLC or mass spectrometry to assess processivity and product specificity.

How does the cis/trans configuration of products from L. plantarum Upp synthetase differ from other bacterial prenyltransferases?

The cis/trans configuration of isoprenoid products is a critical distinguishing feature of different prenyltransferases. L. plantarum Upp synthetase, like other bacterial Upp synthetases, produces undecaprenyl pyrophosphate with a mixed trans,cis-isoprenoid chain configuration. This differs significantly from other bacterial prenyltransferases such as octaprenyl pyrophosphate synthetase (Opp synthetase), which generates products with all-trans configuration.

The specific structural features that determine the cis/trans configuration include:

• The presence of specific "cis-recognition" residues in the active site that influence the geometry of IPP addition to the growing chain.

• Variations in the flexible loop regions that participate in substrate binding and product release.

• Different metal ion coordination geometries that affect the orientation of the substrates during catalysis.

The trans,cis-configuration is essential for the biological function of UPP as a lipid carrier in cell wall biosynthesis, as it provides the appropriate physicochemical properties for integration into the membrane and interaction with cell wall biosynthetic enzymes.

What molecular mechanisms determine the chain length specificity of L. plantarum Upp synthetase?

The chain length specificity of L. plantarum Upp synthetase is determined by several molecular mechanisms:

• Active site architecture: The size and shape of the hydrophobic binding pocket accommodates a specific number of isoprenoid units before steric hindrance prevents further chain elongation.

• Key residues at the bottom of the binding pocket: These residues act as a "molecular ruler" that limits the maximum length of the growing isoprenoid chain.

• Product release mechanism: The enzyme may undergo conformational changes after reaching a specific chain length, facilitating product release.

• Hydrophobic interactions: The interaction between the growing hydrophobic chain and specific residues lining the active site tunnel influences when the product is released.

• Allosteric regulation: Binding of the growing product to secondary sites on the enzyme may trigger conformational changes that reduce catalytic efficiency after reaching the optimal chain length.

Research using site-directed mutagenesis of conserved residues in the hydrophobic binding pocket can help identify the specific amino acids responsible for chain length determination in L. plantarum Upp synthetase. Comparison with homologous enzymes producing different chain lengths (e.g., Fpp synthetase or Opp synthetase) can provide insights into the structural determinants of chain length specificity.

How does substrate binding affect the conformational dynamics of L. plantarum Upp synthetase?

Substrate binding induces significant conformational changes in L. plantarum Upp synthetase that are essential for its catalytic function:

• Induced-fit mechanism: Binding of the allylic substrate (FPP) triggers the first conformational change, creating a binding pocket for IPP.

• Metal ion coordination: Mg²⁺ ions form coordination complexes with the pyrophosphate moieties of both substrates, properly orienting them for the condensation reaction.

• Dynamic changes during catalysis:

  • Closure of flexible loops over the active site upon substrate binding

  • Rearrangement of catalytic residues to optimize distances for reaction chemistry

  • Sequential opening and closing of the active site during multiple rounds of IPP addition

• Product accommodation: As the isoprenoid chain grows, the enzyme undergoes progressive conformational adjustments to accommodate the increasing chain length.

• Triton X-100 effect: The detergent requirement suggests that amphipathic molecules may help stabilize certain conformational states of the enzyme, particularly those involved in binding or releasing the hydrophobic isoprenoid products.

Advanced biophysical methods such as hydrogen-deuterium exchange mass spectrometry (HDX-MS), fluorescence resonance energy transfer (FRET), and molecular dynamics simulations can provide detailed insights into these conformational dynamics and their relationship to the catalytic cycle.

What structural features of L. plantarum Upp synthetase determine its catalytic mechanism?

The catalytic mechanism of L. plantarum Upp synthetase is determined by several key structural features:

• Active site architecture: Contains a conserved catalytic region with specific aspartate-rich motifs (typically DDXXD) that coordinate essential Mg²⁺ ions required for substrate binding and catalysis.

• Hydrophobic binding tunnel: A long, hydrophobic tunnel accommodates the growing isoprenoid chain during sequential condensation reactions.

• Substrate binding sites:

  • FPP binding site: Binds the allylic substrate through interactions with the pyrophosphate group and the isoprenoid chain

  • IPP binding site: Positioned to facilitate nucleophilic attack on the allylic substrate

• Conserved basic residues: Typically arginine or lysine residues that stabilize the developing negative charge during the reaction and participate in pyrophosphate release.

• Flexible loop regions: These undergo conformational changes during catalysis, controlling substrate access and product release.

How do mutations in conserved residues affect the activity and specificity of L. plantarum Upp synthetase?

Mutations in conserved residues of L. plantarum Upp synthetase can significantly impact enzyme activity and specificity in several ways:

Systematic site-directed mutagenesis studies combined with kinetic analysis, product characterization, and structural investigations can provide valuable insights into the structure-function relationships of L. plantarum Upp synthetase. These studies are particularly valuable for rational enzyme engineering efforts aimed at modifying substrate specificity or improving catalytic efficiency.

What is the role of oligomerization in L. plantarum Upp synthetase function?

Oligomerization plays a significant role in the function of L. plantarum Upp synthetase:

• Quaternary structure: Upp synthetases typically exist as homodimers or higher-order oligomers in solution, with the oligomeric state influencing catalytic activity.

• Subunit interactions: The interface between subunits often contains residues that contribute to the complete active site, making oligomerization essential for full catalytic function.

• Allosteric regulation: Communication between subunits can facilitate allosteric regulation, where substrate binding at one active site influences the activity of adjacent active sites.

• Stabilization effects:

  • Oligomerization often enhances thermal stability

  • Protects hydrophobic surfaces from inappropriate interactions

  • Increases resistance to proteolytic degradation

• Membrane interaction: The oligomeric structure may facilitate optimal interaction with the membrane, which is relevant given the membrane-associated nature of the enzyme's product (UPP) and the enzyme's detergent dependence.

Analytical techniques such as size exclusion chromatography, analytical ultracentrifugation, native PAGE, and crosslinking studies can be used to characterize the oligomeric state of recombinant L. plantarum Upp synthetase and its relationship to catalytic activity. Disrupting oligomerization through mutation of interface residues can provide insights into the functional significance of quaternary structure.

What compounds effectively inhibit L. plantarum Upp synthetase activity and what are their mechanisms of action?

Several classes of compounds can inhibit L. plantarum Upp synthetase through distinct mechanisms of action:

• Substrate analogs:

  • FPP analogs with modified pyrophosphate groups

  • IPP analogs with altered double bonds or phosphate groups

  • Mechanism: Competitive inhibition by binding to substrate binding sites

• Product analogs:

  • Undecaprenyl monophosphate or dephosphorylated derivatives

  • Mechanism: Product inhibition and feedback regulation

• Bisphosphonates:

  • Compounds containing P-C-P linkages that mimic pyrophosphate

  • Mechanism: Coordination with catalytic Mg²⁺ ions, preventing proper substrate binding

• Metal chelators:

  • EDTA, EGTA, and similar compounds

  • Mechanism: Sequestration of essential Mg²⁺ ions required for catalysis

• Cationic peptides:

  • Positively charged peptides that interact with pyrophosphate binding sites

  • Mechanism: Electrostatic interference with substrate binding

• Detergent-like inhibitors:

  • Cationic or non-ionic surfactants that disrupt the enzyme-detergent-substrate interactions

  • Mechanism: Alteration of the microenvironment required for optimal activity

Inhibitor studies not only provide insights into the catalytic mechanism of L. plantarum Upp synthetase but may also have potential applications in the development of novel antimicrobial agents, as Upp synthetase is essential for bacterial cell wall biosynthesis.

How is the expression and activity of L. plantarum Upp synthetase regulated in vivo?

The expression and activity of L. plantarum Upp synthetase is regulated through multiple mechanisms in vivo:

• Transcriptional regulation:

  • The uppS gene expression is likely coordinated with other genes involved in cell wall biosynthesis

  • Promoter elements may respond to cell wall stress, allowing upregulation during periods of active cell wall synthesis

  • Growth phase-dependent expression patterns, with higher expression during exponential growth

• Post-translational modifications:

  • Potential phosphorylation sites may modulate enzyme activity

  • Redox-sensitive cysteine residues could respond to oxidative stress

• Allosteric regulation:

  • Feedback inhibition by undecaprenyl pyrophosphate or downstream metabolites

  • Small molecule effectors that signal cellular energy status

• Spatial regulation:

  • Localization to specific regions of the cell membrane where cell wall synthesis is active

  • Interaction with other enzymes in the peptidoglycan biosynthesis pathway to form functional complexes

• Protein-protein interactions:

  • Associations with other enzymes in the cell wall biosynthetic pathway

  • Potential interactions with regulatory proteins that modulate activity

Research approaches to study in vivo regulation include reporter gene fusions, quantitative proteomics, metabolic labeling, and the creation of conditional expression strains to monitor the physiological consequences of altered Upp synthetase levels.

What are the effects of environmental factors on L. plantarum Upp synthetase stability and activity?

Environmental factors significantly influence the stability and activity of L. plantarum Upp synthetase:

Understanding these environmental influences is crucial for designing robust experimental procedures and interpreting results accurately. For long-term storage, the enzyme is typically most stable when stored at -80°C in buffer containing glycerol (20-50%), detergent, and reducing agent.

How can recombinant L. plantarum Upp synthetase be utilized for the biosynthesis of novel isoprenoid compounds?

Recombinant L. plantarum Upp synthetase offers several opportunities for the biosynthesis of novel isoprenoid compounds:

• Engineered substrate specificity:

  • Site-directed mutagenesis of active site residues can alter substrate preferences

  • Creation of enzymes that accept non-natural allylic substrates with modified isoprenoid chains

  • Engineering variants that incorporate functionalized IPP analogs

• Controlled chain length products:

  • Mutations in the hydrophobic binding pocket can generate enzymes producing shorter or longer isoprenoid chains

  • Development of variants with relaxed chain length specificity for the production of diverse polyprenyl compounds

• Modified stereochemistry:

  • Engineering variants that alter the cis/trans configuration of isoprenoid products

  • Creation of enzymes that produce all-cis or specifically patterned cis/trans products

• Biocatalytic applications:

  • Use in chemoenzymatic synthesis of complex isoprenoid compounds

  • One-pot enzymatic cascades combining multiple prenyltransferases

  • Immobilized enzyme technology for continuous production processes

• Cell factory development:

  • Integration into metabolically engineered microorganisms for the production of valuable isoprenoids

  • Coupling with engineered IPP/DMAPP biosynthetic pathways

These applications require detailed understanding of structure-function relationships and often benefit from directed evolution approaches to develop enzymes with desired catalytic properties.

What experimental approaches are most effective for studying the in vivo function of L. plantarum Upp synthetase?

Several complementary experimental approaches can effectively elucidate the in vivo function of L. plantarum Upp synthetase:

• Genetic manipulation techniques:

  • Conditional expression systems to control uppS expression levels

  • CRISPR-Cas9 genome editing for precise genetic modifications

  • Regulatable gene disruption systems to study essentiality under various conditions

• Functional complementation studies:

  • Expression of L. plantarum uppS in heterologous hosts with temperature-sensitive or depleted native uppS

  • Cross-species complementation to assess functional conservation

  • Rescue experiments with mutant variants to identify critical residues for in vivo function

• In vivo labeling approaches:

  • Metabolic labeling with isotope-labeled precursors (¹³C-mevalonate or ¹⁴C-IPP)

  • Click chemistry with azide/alkyne-modified isoprenoid precursors

  • Pulse-chase experiments to measure UPP turnover rates

• Microscopy and localization studies:

  • Fluorescent protein fusions to track subcellular localization

  • Super-resolution microscopy to visualize co-localization with cell wall synthesis machinery

  • Time-lapse microscopy to monitor dynamics during cell division

• Systems biology approaches:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to identify interaction partners and post-translational modifications

  • Metabolomics to detect changes in isoprenoid and cell wall precursor pools

These approaches can reveal how Upp synthetase integrates into the broader cellular context of cell wall biosynthesis and identify potential regulatory mechanisms that control its activity in response to changing cellular conditions.

What are the implications of uppS essentiality for antimicrobial drug development?

The essentiality of uppS across bacterial species, including L. plantarum, has significant implications for antimicrobial drug development:

• Target validation:

  • The demonstration that uppS is essential for growth in S. pneumoniae R6, as shown through regulatable gene disruption systems, provides strong validation for targeting this enzyme in antimicrobial development.

  • Similar essentiality is likely in L. plantarum and other bacterial pathogens, making Upp synthetase a broadly applicable target.

• Advantages as a drug target:

  • High conservation allows for broad-spectrum activity

  • No human homologs with high similarity reduces the risk of off-target effects

  • Involvement in a pathway (cell wall synthesis) with a proven track record for successful antibiotics

  • Biochemical assays amenable to high-throughput screening

• Rational drug design approaches:

  • Structure-based design utilizing crystallographic data from bacterial Upp synthetases

  • Fragment-based lead discovery targeting the active site or allosteric sites

  • Peptide inhibitors designed to disrupt oligomerization or membrane association

• Resistance considerations:

  • The essential nature and high conservation of uppS suggests a high genetic barrier to resistance

  • Potential resistance mechanisms include target overexpression, efflux pump upregulation, or modification of cell envelope permeability

  • Combination therapy approaches could mitigate resistance development

• Delivery strategies:

  • Small molecule inhibitors targeting the active site

  • Peptidomimetics targeting protein-protein interfaces

  • Prodrug approaches to enhance bacterial penetration

Development of Upp synthetase inhibitors represents a promising avenue for novel antimicrobial agents, particularly given the continuing challenge of antimicrobial resistance and the limited pipeline of new antibiotics with novel mechanisms of action.