Recombinant Idiomarina loihiensis UDP-N-acetylenolpyruvoylglucosamine reductase (murB)

What is the role of MurB in bacterial cell wall biosynthesis?

MurB (UDP-N-acetylenolpyruvoylglucosamine reductase) catalyzes the second step in the peptidoglycan biosynthesis pathway, specifically the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UDP-MurNAc). This reaction is part of the classical de novo biosynthesis pathway that begins with UDP-GlcNAc . The peptidoglycan cell wall is crucial for bacterial survival, providing structural integrity and protection against osmotic pressure. In I. loihiensis, this pathway is particularly important as genomic studies have revealed that this organism relies primarily on amino acid catabolism rather than sugar fermentation for carbon and energy .

What structural and functional features characterize the MurB enzyme from I. loihiensis?

The MurB enzyme from I. loihiensis shares structural and functional similarities with other bacterial MurB enzymes. As a UDP-N-acetylenolpyruvoylglucosamine reductase, it contains binding sites for its substrate (UDP-N-acetylenolpyruvylglucosamine) and cofactor (NADPH). While specific structural information for I. loihiensis MurB is limited in the search results, studies of related MurB enzymes indicate that they typically feature a multi-domain structure with N-terminal and C-terminal domains connected by a flexible linker region. The enzyme catalyzes a redox reaction where NADPH provides reducing power to convert the enolpyruvyl moiety to a lactyl group . The enzyme plays a critical role in peptidoglycan biosynthesis, which is essential for bacterial cell wall integrity and serves as a target for antibiotic development.

What is the recommended protocol for heterologous expression and purification of recombinant I. loihiensis MurB?

Based on studies with related MurB enzymes and other recombinant proteins from I. loihiensis, the following protocol is recommended:

• Gene Cloning: Amplify the MurB gene from I. loihiensis genomic DNA using PCR with specific primers containing appropriate restriction sites. Clone the gene into an expression vector (e.g., pET series) with a C-terminal His6-tag for purification purposes .

• Expression System: Transform the construct into an E. coli expression strain (e.g., BL21(DE3)). For MurB enzymes, expression in E. coli has been shown to be efficient .

• Culture Conditions: Grow transformed cells in LB medium supplemented with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8. Induce protein expression with ISOPROPYL β-D-1-thiogalactopyranoside (IPTG, typically 0.5-1 mM) and continue incubation at a reduced temperature (16-22°C) for 16-18 hours to enhance protein solubility.

• Cell Harvesting and Lysis: Harvest cells by centrifugation and resuspend in lysis buffer containing appropriate protease inhibitors. Lyse cells using sonication or alternative methods.

• Purification: Purify the His-tagged protein using Ni²⁺ affinity chromatography followed by gel filtration chromatography to ensure high purity . For long-term stability, add glycerol (approximately 20%) to the final protein solution before storage at -80°C .

This approach has been successful for related enzymes and should be adaptable to I. loihiensis MurB based on the characteristics of this marine bacterium.

How can researchers optimize the activity assay for recombinant I. loihiensis MurB?

The optimization of the MurB activity assay should consider several parameters:

• Buffer Selection: Test various buffers at different pH values. For Mur enzymes, HEPES buffer at pH 7.0 has been found optimal for some assays, while Bis-tris propane at pH 7.0 has shown compatibility across multiple Mur enzymes .

• Temperature and pH Optimization: Determine the optimal temperature and pH for the enzyme activity. Studies with related enzymes suggest testing a temperature range of 22-45°C and pH range of 6-9. Note that while activity might be higher at elevated temperatures (e.g., 45°C), protein stability may decrease rapidly .

• Metal Ion Requirement: Assess the effect of divalent metal ions (particularly Mg²⁺) on enzyme activity. Addition of 10 mM MgCl₂ has been shown to significantly enhance reaction rates for some phosphatase enzymes .

• Kinetic Measurement: MurB activity can be measured kinetically by following the oxidation of NADPH spectrophotometrically at 340 nm. Initially measure activity for 5-10 minutes to determine the linear range of the reaction .

• Reaction Components: The complete reaction mixture should contain:

  • Recombinant MurB enzyme

  • UDP-N-acetylenolpyruvylglucosamine substrate

  • NADPH cofactor

  • Appropriate buffer system

  • MgCl₂ or other required metal ions

• Data Analysis: Calculate enzyme activity in terms of the rate of NADPH oxidation. Determine key kinetic parameters (Km, Vmax, kcat) by varying substrate concentrations and fitting data to appropriate enzyme kinetic models.

This methodological approach will help ensure reliable and reproducible assessment of MurB activity.

What techniques are most effective for characterizing the substrate specificity of I. loihiensis MurB?

To thoroughly characterize the substrate specificity of I. loihiensis MurB, a multi-faceted approach is recommended:

• Kinetic Analysis with Structural Analogs: Test the enzyme with the natural substrate (UDP-N-acetylenolpyruvylglucosamine) and structural analogs to determine the structural requirements for substrate recognition. Calculate kinetic parameters (Km, kcat, kcat/Km) for each substrate to quantify specificity.

• Mass Spectrometry-Based Assays: Employ mass spectrometry to identify and quantify reaction products. Tools such as mMASS can be used for data analysis . This approach allows precise identification of the products formed with different substrates.

• Isothermal Titration Calorimetry (ITC): Use ITC to measure binding affinities of different substrates to the enzyme, providing thermodynamic parameters of the interaction.

• Spectroscopic Methods: Monitor changes in intrinsic fluorescence or circular dichroism upon substrate binding to assess structural changes associated with substrate recognition.

• Computational Approaches: Implement molecular docking and molecular dynamics simulations to predict interactions between the enzyme and various potential substrates . This can guide experimental design and help interpret experimental results.

• Site-Directed Mutagenesis: Identify and mutate key residues predicted to be involved in substrate binding or catalysis, then evaluate the effect on substrate specificity and catalytic efficiency.

By combining these approaches, researchers can obtain a comprehensive understanding of the substrate specificity of I. loihiensis MurB, which is essential for both fundamental enzymology and potential biotechnological applications.

How does the environmental adaptation of I. loihiensis affect the properties of its MurB enzyme compared to mesophilic counterparts?

I. loihiensis was isolated from hydrothermal vents on the Lōihi Seamount, Hawaii, and exhibits remarkable adaptability to a wide range of temperatures (4°C to 46°C) and salinities (0.5% to 20% NaCl) . These environmental adaptations likely influence the properties of its enzymes, including MurB:

• Temperature Stability: The MurB enzyme from I. loihiensis might exhibit broader temperature stability compared to mesophilic counterparts, reflecting the organism's capacity to survive in environments with temperature fluctuations. Research on related enzymes has shown that optimal activity can occur at different temperatures, with significant variation in stability profiles .

• Salt Tolerance: Given the halophilic nature of I. loihiensis, its MurB enzyme likely possesses structural features that enable stability and activity in high-salt environments. These adaptations typically include an increased proportion of acidic amino acids on the protein surface, altered hydrophobic interactions, and specific ion-binding sites.

• pH Adaptability: The enzyme may show activity across a broader pH range compared to mesophilic counterparts. Studies with related enzymes have demonstrated activity across pH 6-9, with retention of significant activity even at pH 4 and pH 10 .

• Cofactor Requirements: Environmental adaptations might influence the enzyme's interaction with cofactors like NADPH. For instance, the binding affinity or the rate of cofactor oxidation could differ from mesophilic enzymes to accommodate the unique cellular environment of I. loihiensis.

• Structural Stability: Comparative analysis might reveal structural differences that contribute to the enzyme's stability under extreme conditions, such as additional salt bridges, disulfide bonds, or altered surface charge distribution.

To systematically investigate these differences, researchers should conduct comparative biochemical and structural studies of MurB from I. loihiensis and mesophilic bacteria under varying conditions of temperature, salinity, and pH.

What role does MurB play in the intrinsic antibiotic resistance mechanisms of I. loihiensis?

The MurB enzyme and the peptidoglycan biosynthesis pathway contribute to antibiotic resistance in I. loihiensis through several mechanisms:

• Alternative Pathway Compensation: I. loihiensis and related bacteria can utilize an anabolic recycling route that bypasses the classical de novo biosynthesis of UDP-MurNAc . This alternative pathway can potentially compensate for inhibition of MurB by antibiotics.

• Fosfomycin Resistance: While fosfomycin typically targets MurA in the peptidoglycan biosynthesis pathway, the functionality of MurB and the entire pathway influences susceptibility. In Pseudomonas putida, disruption of genes in the anabolic recycling pathway leads to fosfomycin hypersensitivity . Genomic analysis has revealed that I. loihiensis L2TR contains genes conferring intrinsic resistance to fosfomycin, including murA .

• Impact on β-lactam Resistance: In P. aeruginosa, disruption of the peptidoglycan recycling pathway causes elevated expression of AmpC β-lactamase, increasing resistance to β-lactam antibiotics. This effect was linked to reduced steady-state levels of UDP-MurNAc-pentapeptide . Similar mechanisms may be present in I. loihiensis.

• Metabolic Pool Regulation: The activity of MurB affects the intracellular levels of UDP-MurNAc and related metabolites. Alterations in these pools can influence sensitivity to antibiotics targeting cell wall synthesis .

• Genomic Islands and Horizontal Gene Transfer: Genomic analysis of I. loihiensis revealed the presence of genomic islands that may contain antibiotic resistance genes . These islands can facilitate the spread of resistance mechanisms, potentially affecting the function or regulation of cell wall synthesis enzymes including MurB.

Understanding these resistance mechanisms is crucial for researchers developing strategies to target MurB or related enzymes for antimicrobial development.

How can structural data be leveraged for rational design of inhibitors targeting I. loihiensis MurB?

Rational design of inhibitors targeting I. loihiensis MurB can be approached through a structural biology-based strategy:

• Structure Determination: Obtain high-resolution crystal structures of I. loihiensis MurB, both in apo form and in complex with substrates, products, and known inhibitors. If direct structural determination is challenging, create homology models based on closely related MurB enzymes.

• Active Site Mapping: Identify the key residues involved in substrate binding and catalysis through structural analysis and site-directed mutagenesis. Focus particularly on:

  • NADPH binding pocket

  • UDP-N-acetylenolpyruvylglucosamine binding site

  • Catalytic residues involved in the reduction reaction

• Virtual Screening: Implement computational approaches for inhibitor discovery:

  • Structure-based virtual screening of compound libraries (e.g., ChemSpider, DrugBank, Zinc database)

  • Molecular docking to evaluate binding affinities and poses

  • Use programs like AutoDock Vina with optimized parameters: population size of 150, maximum of 27K generations, and maximum generation evaluation of 2,500K

  • Apply a binding affinity threshold (e.g., -9.0 Kcal/mol) for initial compound selection

• Molecular Dynamics Simulations: Evaluate the stability of protein-ligand complexes through:

  • Production of complexes in 10 Å × 10 Å × 10 Å gradient boxes

  • Implementation of TIP4P transferable intermolecular potential for water molecules

  • Use of appropriate force fields (e.g., OPLS_2005)

  • Simulations for 100 ns intervals in triplicate to ensure reproducibility

• Validation and Refinement:

  • Synthesis or acquisition of computationally identified hit compounds

  • Biochemical assays to determine inhibitory potency (IC50, Ki values)

  • Structure-activity relationship studies to optimize lead compounds

  • Co-crystallization of promising inhibitors with MurB to guide further optimization

• Specificity Analysis: Evaluate selectivity by testing inhibitors against human enzymes with similar functions or structures to minimize potential side effects.

This integrated approach can facilitate the development of potent and selective inhibitors of I. loihiensis MurB with potential antimicrobial applications.

What are the main challenges in expressing and purifying active recombinant I. loihiensis MurB, and how can they be overcome?

Several challenges may arise when working with recombinant I. loihiensis MurB:

• Protein Solubility Issues:

  • Challenge: MurB may form inclusion bodies during heterologous expression in E. coli.

  • Solution: Optimize expression conditions by lowering induction temperature (16-22°C), using lower IPTG concentrations (0.1-0.5 mM), or employing specialized E. coli strains (e.g., Arctic Express, Rosetta). Alternatively, consider fusion tags like MBP (maltose-binding protein) or SUMO to enhance solubility.

• Protein Stability Concerns:

  • Challenge: As observed with related enzymes, MurB activity can decrease significantly at higher temperatures (e.g., 70% loss at 37°C within 40 minutes) .

  • Solution: Include stabilizers like glycerol (20%) in storage buffers and work at lower temperatures. For kinetic experiments with temperature-sensitive enzymes, reduce reaction times (e.g., to 2.5 minutes) to minimize activity loss during assays .

• Cofactor Binding and Activity:

  • Challenge: Ensuring proper cofactor (NADPH) binding and activity in the recombinant enzyme.

  • Solution: Supplement reaction buffers with appropriate concentrations of NADPH and additional cofactors like Mg²⁺ (e.g., 10 mM MgCl₂), which has been shown to enhance activity of related enzymes by over 16-fold .

• Enzyme Assay Detection Limits:

  • Challenge: Developing sensitive assays to measure MurB activity, especially if expression yields are low.

  • Solution: Implement multiple detection methods including UV-Vis spectroscopy for NADPH oxidation, mass spectrometry for product formation, and fluorescence-based assays for enhanced sensitivity.

• Marine Organism Adaptation:

  • Challenge: I. loihiensis is adapted to marine environments with high salt concentrations, which may affect protein folding and activity when expressed in standard systems.

  • Solution: Consider including NaCl or other salts in purification and assay buffers to mimic the native environment of I. loihiensis. Testing a range of salt concentrations (0.5-5% NaCl) may help identify optimal conditions for enzyme activity and stability.

By addressing these challenges systematically, researchers can successfully express and purify active recombinant I. loihiensis MurB for subsequent biochemical and structural studies.

How can researchers address the potential interference of metals and other ions in MurB activity assays for I. loihiensis?

Metal ions and other ionic species can significantly influence MurB activity and assay results:

• Identification of Metal Dependencies:

  • Approach: Systematically screen various metal ions (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺, Fe²⁺) at different concentrations (1-20 mM) to identify specific requirements or inhibitory effects.

  • Methodology: Compare enzyme activity with and without EDTA treatment to remove bound metals, followed by reconstitution with specific metal ions.

• Background Electrolyte Effects:

  • Challenge: The nature of the background electrolyte can influence enzyme activity, as demonstrated in studies with I. loihiensis where interactions with uranium differed between seawater and NaClO₄ solutions .

  • Solution: Test multiple background electrolytes (e.g., NaCl, KCl, NaClO₄) to identify optimal conditions for MurB activity. Consider the native marine environment of I. loihiensis when selecting appropriate salt compositions.

• pH-Dependent Metal Binding:

  • Issue: Metal-enzyme interactions can vary with pH, affecting enzyme structure and function .

  • Approach: Evaluate metal effects across a range of pH values (pH 5-9) to identify optimal conditions for activity and to understand how pH influences metal-enzyme interactions.

• Buffer Compatibility:

  • Strategy: Select buffers that minimize metal precipitation or complex formation. Phosphate buffers may be problematic due to precipitation with certain metals, while HEPES or Bis-tris propane buffers may offer better compatibility .

  • Validation: Include appropriate controls with and without metals in each buffer system to confirm buffer compatibility.

• Methodological Controls and Standards:

  • Implementation: Include internal standards in assays to normalize for metal-induced variations.

  • Data Analysis: Apply statistical methods to distinguish true enzyme activity changes from metal-induced assay artifacts.

• Advanced Spectroscopic Approaches:

  • Techniques: Employ methods like Electron Paramagnetic Resonance (EPR) or X-ray Absorption Spectroscopy (XAS) to characterize metal binding sites and their effects on enzyme structure.

  • Application: These approaches can provide atomic-level insights into how metals interact with MurB and influence its catalytic mechanism.

By systematically addressing these factors, researchers can develop robust assays for I. loihiensis MurB that account for metal and ion effects, ensuring reliable and reproducible activity measurements.

What are the key considerations for storage and maintaining long-term stability of purified recombinant I. loihiensis MurB?

Ensuring the long-term stability of purified recombinant I. loihiensis MurB requires careful attention to storage conditions and buffer composition:

• Temperature Optimization:

  • Short-term Storage: Store at 4°C for immediate use (typically stable for 24-48 hours).

  • Long-term Storage: Store at -80°C in small aliquots to avoid repeated freeze-thaw cycles. Studies with related enzymes have shown that storage at -80°C with appropriate additives can maintain stability for extended periods .

  • Freeze-thaw Impact: Evaluate enzyme activity after multiple freeze-thaw cycles to determine stability limits and establish proper aliquoting protocols.

• Buffer Composition:

  • Glycerol Addition: Include 20% glycerol as a cryoprotectant for -80°C storage, as this has been shown to increase long-term stability of similar enzymes .

  • pH Selection: Maintain pH within the stability range of the enzyme (typically pH 7-8 for many proteins, but may require optimization for I. loihiensis MurB).

  • Salt Concentration: Consider including NaCl at concentrations that mimic the natural marine environment of I. loihiensis (0.5-3% may be appropriate).

• Protective Additives:

  • Reducing Agents: Include reducing agents like DTT (1-5 mM) or β-mercaptoethanol (5-10 mM) to prevent oxidation of cysteine residues.

  • Protease Inhibitors: Add a protease inhibitor cocktail to prevent degradation by trace proteases.

  • Metal Chelators: Consider EDTA (0.1-1 mM) to prevent metal-catalyzed oxidation, but evaluate its impact on enzyme activity if metals are required for function.

• Protein Concentration Considerations:

  • Optimal Range: Determine the concentration range that maintains stability (typically 0.5-5 mg/mL for many proteins).

  • Preventing Aggregation: For proteins prone to aggregation, lower concentrations may be preferable, or consider additives like non-ionic detergents (e.g., 0.01% Triton X-100) or osmolytes (e.g., trehalose).

• Stability Monitoring:

  • Activity Assays: Regularly measure enzyme activity to track stability over time.

  • Physical Methods: Employ techniques like dynamic light scattering or size-exclusion chromatography to monitor aggregation state.

  • Thermal Shift Assays: Use differential scanning fluorimetry to identify stabilizing conditions and additives.

• Lyophilization Potential:

  • Feasibility Testing: Evaluate if lyophilization (freeze-drying) with appropriate lyoprotectants (e.g., sucrose, trehalose) can provide enhanced long-term stability.

  • Reconstitution Protocol: If lyophilization is viable, develop and validate a reliable reconstitution protocol.

By systematically optimizing these parameters, researchers can maximize the stability and shelf-life of purified recombinant I. loihiensis MurB, ensuring consistent results across experiments.

How does I. loihiensis MurB compare structurally and functionally to MurB enzymes from other extremophilic and mesophilic bacteria?

A comparative analysis of MurB enzymes from various sources reveals important adaptations and functional differences:

• Sequence Homology and Phylogenetic Relationships:

  • I. loihiensis MurB likely shares sequence similarity with other γ-proteobacterial MurB enzymes, particularly those from marine or extremophilic sources.

  • Phylogenetic analysis can place I. loihiensis MurB in context with enzymes from both extremophiles and mesophiles, revealing evolutionary adaptations to different environments.

• Structural Adaptations to Marine/Hydrothermal Environments:

  • Salt Tolerance: Compared to mesophilic counterparts, I. loihiensis MurB may contain more acidic surface residues, altered ion-binding sites, and specific salt-bridge patterns that enable function in high-salt environments.

  • Temperature Adaptations: The enzyme likely possesses structural features allowing functionality across the wide temperature range that I. loihiensis can tolerate (4-46°C) .

  • Pressure Considerations: Given its deep-sea origin, structural adaptations for high-pressure environments may be present, potentially including more compact hydrophobic cores or specific volume-change minimizing features.

• Catalytic Efficiency Parameters:

  Bacterial Source	kcat (s⁻¹)	Km (μM)	kcat/Km (M⁻¹s⁻¹)	Optimal Temp (°C)	pH Optimum	Salt Tolerance
  I. loihiensis (marine/extremophile)	Predicted moderate-high	Predicted moderate	Predicted moderate-high	Expected 30-45	Expected 7-8	Expected high
  E. coli (mesophile)	100-350	10-50	10⁶-10⁷	30-37	7.0-7.5	Moderate
  M. tuberculosis (pathogen)	20-80	20-100	10⁵-10⁶	37	6.5-7.5	Low
  Thermophilic bacteria	50-200	5-50	10⁶-10⁷	50-70	7.5-8.5	Variable

  Note: Specific values for I. loihiensis MurB are predicted based on typical patterns observed between extremophilic and mesophilic enzymes, as exact kinetic parameters were not provided in the search results.

• Cofactor Preferences and Binding:

  • NADPH Binding: I. loihiensis MurB likely utilizes NADPH as a cofactor, similar to other MurB enzymes, but may show different binding affinities or kinetics compared to mesophilic counterparts.

  • Metal Requirements: Given the high metal concentrations in hydrothermal environments, I. loihiensis MurB may exhibit altered metal ion preferences or binding sites compared to mesophilic enzymes .

• Substrate Specificity:

  • While the primary function of converting UDP-N-acetylenolpyruvylglucosamine to UDP-MurNAc is conserved, the enzyme may show different substrate promiscuity compared to mesophilic counterparts.

  • This could be investigated by testing activity with substrate analogs and comparing specificity constants across different bacterial sources.

Understanding these comparative aspects can provide insights into enzymatic adaptations to extreme environments and guide biotechnological applications of I. loihiensis MurB.

What potential biotechnological applications exist for recombinant I. loihiensis MurB beyond antimicrobial development?

Recombinant I. loihiensis MurB offers several promising biotechnological applications beyond its role as an antimicrobial target:

• Bioremediation of Heavy Metals and Radionuclides:

  • Scientific Basis: Studies have shown that I. loihiensis possesses remarkable abilities to interact with metals including uranium through biosorption and biomineralization . The genus Idiomarina contains various resistance proteins and transporters associated with metal tolerance, including for Fe, Cu, Zn, Pb, and Cd .

  • Application: Engineered MurB or MurB-containing cell wall fragments could be developed as bioadsorbents for heavy metal removal from contaminated environments.

  • Advantage: The enzyme's adaptation to extreme conditions could make it suitable for bioremediation in challenging environments with high salinity or temperature fluctuations.

• Biocatalysis for Specialized Carbohydrate Synthesis:

  • Potential: The reductive capability of MurB could be harnessed for stereoselective reduction of α,β-unsaturated carbonyl compounds in carbohydrate synthesis.

  • Target Applications: Production of specialized sugars for glycobiology research, pharmaceutical intermediates, or rare sugar derivatives.

  • Advantage: The enzyme's potential stability in high-salt conditions could enable unique reaction environments not tolerated by conventional biocatalysts.

• Biomaterial Development:

  • Concept: The cell wall biosynthesis role of MurB could be leveraged for creating novel biomaterials.

  • Applications: Development of peptidoglycan-mimetic polymers for drug delivery systems, tissue engineering scaffolds, or biodegradable plastics.

  • Approach: Engineering MurB to accept modified substrates could lead to novel polymer materials with tailored properties.

• Biosensors for Environmental Monitoring:

  • Design: MurB activity is linked to NADPH oxidation, which can be monitored spectrophotometrically. This property can be utilized to develop biosensors.

  • Applications: Detection of specific inhibitors, heavy metals, or environmental toxicants that affect enzyme activity.

  • Advantage: The enzyme's extremophilic nature could enable biosensor functionality in harsh environments where conventional proteins would denature.

• Protein Engineering Platform:

  • Approach: The unique adaptations of I. loihiensis MurB to extreme conditions make it an excellent scaffold for protein engineering studies.

  • Applications: Development of proteins with enhanced thermostability, halotolerance, or novel catalytic properties.

  • Research Value: Studying the structure-function relationships in this enzyme could provide valuable insights for rational design of enzymes with improved industrial properties.

• Educational and Research Tools:

  • Usage: Well-characterized enzymes like MurB can serve as model systems for teaching and research in enzymology, protein structure, and drug design.

  • Applications: Development of laboratory kits for educational purposes or standardized research tools for comparing enzymatic properties.

These diverse applications highlight the potential value of recombinant I. loihiensis MurB beyond its traditional role in bacterial cell wall synthesis and as an antimicrobial target.

How might genomic and proteomic data from I. loihiensis inform novel pathway engineering approaches involving MurB?

Genomic and proteomic data from I. loihiensis can inspire innovative pathway engineering strategies:

• Alternative Peptidoglycan Recycling Pathways:

  • Genomic Insight: Analysis has revealed that many bacteria, unlike E. coli, utilize a cell wall salvage pathway that contributes to the pool of UDP-MurNAc and confers intrinsic resistance to fosfomycin .

  • Engineering Opportunity: This alternative pathway, involving enzymes like MupP (MurNAc 6-phosphate phosphatase), could be integrated with MurB to create novel cell wall biosynthesis routes in heterologous hosts.

  • Application: Development of engineered bacteria with enhanced antibiotic resistance profiles or modified cell wall properties for industrial applications.

• Metal-Interaction Pathways:

  • Genomic Basis: Idiomarina species contain numerous genes for metal resistance and transport, including those for Fe, Cu, Zn, Pb, and Cd . Studies have demonstrated I. loihiensis' ability to biomineralize uranium through interactions with cell surface components .

  • Engineering Approach: MurB could be incorporated into engineered pathways that link cell wall synthesis with metal sequestration or biomineralization.

  • Potential Applications: Development of whole-cell biocatalysts for environmental remediation or metal recovery from waste streams.

• Adaptation to Amino Acid Metabolism:

  • Genomic Context: I. loihiensis relies primarily on amino acid catabolism rather than sugar fermentation, with incomplete carbohydrate metabolism pathways but abundant amino acid transport and degradation enzymes .

  • Engineering Strategy: MurB could be integrated into synthetic pathways that link amino acid metabolism directly to cell wall synthesis, potentially bypassing traditional carbon sources.

  • Applications: Creation of microorganisms capable of growth on protein-rich waste streams or in protein-limited environments.

• Stress Response Networks:

  • Proteomic Insight: Comparative genomics has identified core genes related to stress response and salinity tolerance in Idiomarina species .

  • Engineering Opportunity: MurB could be integrated into engineered stress response circuits, where cell wall modification is triggered under specific environmental conditions.

  • Application: Development of biosensors or conditional growth systems for environmental monitoring or controlled bioprocessing.

• Horizontal Gene Transfer Elements:

  • Genomic Feature: Genomic islands have been detected in Idiomarina species, highlighting the role of horizontal gene transfer in acquiring novel genes .

  • Engineering Approach: These genomic islands could inspire the design of synthetic mobile genetic elements for efficient transfer of MurB and related pathway genes between different bacterial species.

  • Application: Facilitated engineering of diverse bacterial species for specialized applications in bioremediation or bioproduction.

• Halophilic Adaptation Mechanisms:

  • Genomic Basis: The genus Idiomarina consists of halophilic and/or haloalkaliphilic organisms with specific adaptations for salt tolerance .

  • Engineering Strategy: MurB and its associated pathways could be integrated with halophilic adaptation mechanisms to engineer salt-tolerant microorganisms.

  • Application: Development of robust biocatalysts for industrial processes in high-salt environments, such as saline wastewater treatment or marine biotechnology.

By leveraging these genomic and proteomic insights, researchers can develop novel pathway engineering approaches that utilize I. loihiensis MurB in contexts beyond its native role, potentially addressing challenges in environmental remediation, industrial biocatalysis, and sustainable manufacturing.

What are the most promising approaches for determining the crystal structure of I. loihiensis MurB?

Determining the crystal structure of I. loihiensis MurB will require a strategic approach addressing potential challenges related to its marine bacterial origin:

By combining these approaches and addressing potential challenges related to the marine origin of I. loihiensis, researchers can maximize the chances of successfully determining the crystal structure of its MurB enzyme.

How might systems biology approaches enhance our understanding of MurB function in the context of I. loihiensis metabolism?

Systems biology approaches can provide comprehensive insights into MurB's role within the broader metabolic network of I. loihiensis:

These systems biology approaches would provide a comprehensive understanding of MurB's role beyond its enzymatic function, revealing its integration within the broader metabolic and regulatory networks of I. loihiensis.

What novel screening approaches could identify potential inhibitors or activators of I. loihiensis MurB for biotechnological applications?

Innovative screening approaches can accelerate the discovery of compounds that modulate I. loihiensis MurB activity:

• High-Throughput Biochemical Assays:

  • Fluorescence-Based Assays: Develop assays monitoring NADPH fluorescence (excitation 340 nm, emission 460 nm) to track MurB activity in real-time with high sensitivity.

  • Coupled Enzyme Assays: Design systems where MurB activity is linked to a reporter enzyme producing a colorimetric or fluorescent signal.

  • Thermal Shift Assays: Screen compounds for their ability to stabilize MurB against thermal denaturation, indicating binding interactions.

  • Automation Integration: Implement robotic liquid handling and plate reading for screening thousands of compounds with minimal reagent consumption.

• Fragment-Based Screening:

  • NMR-Based Screening: Use ligand-observed NMR techniques (STD-NMR, WaterLOGSY) to detect binding of small molecular fragments to MurB.

  • Surface Plasmon Resonance (SPR): Screen fragment libraries for binding to immobilized MurB, providing both binding affinity and kinetic information.

  • Fragment Growing/Linking: Evolve initial fragment hits into more potent compounds through medicinal chemistry optimization.

• Computational Screening:

  • Structure-Based Virtual Screening: As previously described, use molecular docking with optimized parameters (population size of 150, maximum of 27K generations) to screen large compound databases .

  • Pharmacophore Modeling: Develop pharmacophore models based on known MurB substrates and inhibitors to identify compounds with similar functional group arrangements.

  • Quantum Mechanical Approaches: Apply quantum mechanics calculations to understand transition states and design transition state analogs as potential inhibitors.

  • AI-Driven Design: Implement machine learning algorithms trained on existing enzyme modulators to predict novel chemical scaffolds likely to interact with MurB.

• Phenotypic Screening Systems:

  • Reporter Strains: Develop bacterial strains with fluorescent or luminescent reporters linked to cell wall stress responses.

  • Growth-Based Screens: Screen for compounds that synergize with sublethal concentrations of known cell wall inhibitors.

  • Metabolite Profiling: Monitor changes in peptidoglycan precursor levels in response to compound treatment.

• Specialized Screening Approaches:

  • Natural Product Libraries: Screen marine-derived natural products, which may be particularly relevant for a marine bacterium like I. loihiensis.

  • Peptide Display Technologies: Use phage, mRNA, or ribosome display to identify peptides that bind to and modulate MurB activity.

  • DNA-Encoded Libraries: Screen ultra-large DNA-encoded chemical libraries (>10⁹ compounds) for MurB binders.

  • Whole-Cell Activity-Based Protein Profiling: Use activity-based probes that covalently label active MurB in living cells to identify inhibitors that penetrate bacterial membranes.

• Environmentally-Informed Screening:

  • Metal-Compound Complexes: Screen metal-coordinating compounds based on the known interactions of I. loihiensis with metals in its environment .

  • Halophilic Conditions: Conduct screens under high-salt conditions to identify compounds effective in the native-like environment of I. loihiensis.

  • Temperature Gradient Screening: Test compounds across a temperature range (4-46°C) reflecting the adaptability of I. loihiensis .

• Combination Screening Approaches:

  • Matrix Screening: Test compounds in combinations to identify synergistic effects.

  • Sequential Screening Cascade: Implement a funnel approach with increasingly stringent and complex assays to minimize false positives.