Recombinant Citrobacter koseri Thymidylate kinase (tmk)

What is Citrobacter koseri and why is its thymidylate kinase significant for research?

Citrobacter koseri is a gram-negative rod bacterium predominantly associated with infections in immunocompromised individuals and those with significant comorbidities. It is most commonly known to cause urinary tract infections and has developed resistance to multiple conventional antibiotics . Thymidylate kinase (tmk) is an essential enzyme in the bacterial DNA synthesis pathway, catalyzing the phosphorylation of thymidine monophosphate (dTMP) to thymidine diphosphate (dTDP). This critical function makes it an attractive target for antimicrobial drug development, particularly as C. koseri continues to acquire resistance to current antibiotics .

The significance of studying Recombinant C. koseri tmk lies in its potential as a novel drug target. By understanding its structure, function, and inhibition mechanisms, researchers can develop targeted antimicrobials that may circumvent existing resistance mechanisms. Additionally, as a conserved enzyme across bacterial species but with structural differences from human homologs, tmk represents an opportunity for selective targeting of bacterial pathogens.

How is Recombinant Citrobacter koseri Thymidylate kinase expressed and purified for research purposes?

Recombinant C. koseri Thymidylate kinase is typically expressed in Escherichia coli expression systems, as indicated in the product datasheet . The standard protocol involves:

• Cloning: The tmk gene (coding for amino acids 1-213) is amplified from C. koseri genomic DNA (strain ATCC BAA-895/CDC 4225-83/SGSC4696) and cloned into an appropriate expression vector.

• Expression: The recombinant plasmid is transformed into a compatible E. coli strain optimized for protein expression. Expression is typically induced using IPTG or similar inducers under controlled temperature and growth conditions.

• Purification: The expressed protein is purified using affinity chromatography, typically utilizing a tag system that may be determined during the manufacturing process . This is followed by additional purification steps that may include ion exchange chromatography and size exclusion chromatography to achieve >85% purity as verified by SDS-PAGE .

• Storage and Reconstitution: The purified protein can be stored at -20°C, with extended storage recommended at -20°C or -80°C . For optimal stability, the addition of glycerol (typically to a final concentration of 50%) is recommended before aliquoting for long-term storage. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

This process yields research-grade recombinant protein suitable for enzymatic assays, structural studies, inhibitor screening, and other biochemical investigations.

What methodologies are most effective for assessing Citrobacter koseri Thymidylate kinase enzymatic activity?

Several complementary methodologies can be employed to comprehensively assess C. koseri Thymidylate kinase activity:

• Coupled Enzyme Assays: The most common approach involves a coupled enzyme system where ADP production (resulting from tmk activity) is linked to NADH oxidation via pyruvate kinase and lactate dehydrogenase. This allows continuous spectrophotometric monitoring at 340 nm.

• Radiometric Assays: Using radiolabeled substrates (typically [³H]-dTMP or [γ-³²P]-ATP) to directly measure product formation. This provides high sensitivity but requires appropriate radioisotope handling facilities.

• HPLC-Based Methods: Separation and quantification of reaction products (dTDP) using HPLC with UV detection. This method allows direct measurement of product formation without coupling to additional enzymes.

• Malachite Green Phosphate Detection: Measures inorganic phosphate released during the enzymatic reaction, particularly useful when studying phosphorylation mechanisms.

• Fluorescence-Based Assays: Utilizing fluorescent ATP analogs or developing FRET-based sensors to monitor conformational changes during catalysis.

For kinetic parameter determination, researchers should establish optimal buffer conditions (typically containing Mg²⁺ as a cofactor), pH (usually 7.4-8.0), and temperature (37°C for physiological relevance). Michaelis-Menten kinetics should be determined for both substrates (dTMP and ATP), yielding K​m and V​max values that characterize the enzyme's affinity for its substrates and maximum reaction velocity.

A typical reaction buffer might contain:

• 50 mM Tris-HCl (pH 7.5)

• 50 mM KCl

• 5 mM MgCl₂

• 0.1 mg/mL BSA

• 1 mM DTT

• Variable concentrations of substrates (dTMP and ATP)

How does Citrobacter koseri Thymidylate kinase compare structurally and functionally to thymidylate kinases from other bacterial species?

Comparative analysis reveals both conserved features and species-specific variations in thymidylate kinases across bacterial species:

The structural comparison indicates that while C. koseri tmk shares high homology with enterobacterial thymidylate kinases (particularly E. coli), significant differences exist compared to more distant bacterial species. These differences primarily occur in flexible regions and surface loops rather than in the highly conserved active site.

What are the current challenges in developing inhibitors against Citrobacter koseri Thymidylate kinase?

Developing effective inhibitors against C. koseri Thymidylate kinase faces several significant challenges:

• Active Site Conservation: The high conservation of the active site across bacterial species and similarity to human thymidylate kinase makes achieving selectivity challenging. Researchers must identify subtle structural differences that can be exploited for selective targeting.

• Conformational Flexibility: Thymidylate kinases undergo significant conformational changes during catalysis, making structure-based drug design complicated. Multiple protein conformations must be considered when designing potential inhibitors.

• Cell Permeability: Many nucleotide-like inhibitors face challenges in penetrating the gram-negative outer membrane of C. koseri. Compounds must balance potency with appropriate physicochemical properties to ensure bacterial penetration.

• Resistance Development: The potential for rapid resistance development through mutations in the tmk gene must be anticipated. Combination approaches or multi-target inhibitors may be necessary to overcome this challenge.

• Validation Methodologies: Establishing clear correlations between enzymatic inhibition and whole-cell antimicrobial activity requires sophisticated validation methods, including genetic approaches to confirm on-target activity in the bacterial cell.

Innovative approaches to address these challenges include:

• Fragment-based drug discovery to identify novel chemical scaffolds

• Targeting allosteric sites unique to bacterial tmk

• Exploiting differences in conformational dynamics between bacterial and human enzymes

• Developing pro-drug strategies to enhance compound penetration

• Combination approaches targeting multiple steps in the thymidylate synthesis pathway

How can Citrobacter koseri Thymidylate kinase be utilized in structure-based drug design?

Structure-based drug design utilizing C. koseri Thymidylate kinase involves several sophisticated approaches:

• Computational Docking and Virtual Screening: Using the protein sequence to generate homology models (if crystal structures are unavailable) for virtual screening of compound libraries. Molecular docking simulations can identify potential binding modes and interactions of inhibitor candidates.

• Molecular Dynamics Simulations: Investigating protein flexibility and conformational changes during catalysis to identify transient binding pockets that may not be evident in static structures. This approach is particularly valuable for thymidylate kinases, which undergo significant conformational changes during catalysis.

• Fragment-Based Drug Discovery: Screening small molecular fragments for binding to various sites on the enzyme, followed by fragment elaboration or linking to develop high-affinity inhibitors. This approach has proven successful for kinase targets.

• Structure-Activity Relationship Studies: Systematically modifying identified inhibitor scaffolds and correlating structural changes with inhibitory potency to optimize lead compounds. Key parameters to optimize include:

  • Binding affinity to C. koseri tmk

  • Selectivity over human thymidylate kinase

  • Antimicrobial activity against C. koseri

  • Pharmacokinetic properties

• Biophysical Methods for Binding Characterization: Employing techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and thermal shift assays to characterize binding interactions between the enzyme and potential inhibitors.

Successful implementation of these approaches requires access to purified recombinant C. koseri tmk with high purity (>85%) , reliable enzymatic assays, and structural data (either experimental or through homology modeling).

What expression systems and conditions optimize yield and activity of Recombinant Citrobacter koseri Thymidylate kinase?

Optimizing expression of functional Recombinant C. koseri Thymidylate kinase requires careful consideration of multiple parameters:

• Expression Vector Selection:

  • pET-series vectors (particularly pET-28a) are commonly used for high-level expression of recombinant proteins in E. coli

  • The vector should include appropriate tags for purification (His-tag, GST-tag) that don't interfere with enzymatic function

  • Codon optimization for E. coli expression may improve yields

• E. coli Strain Selection:

  • BL21(DE3) and derivatives are most commonly used for recombinant protein expression

  • Rosetta or CodonPlus strains can address codon bias issues

  • Strains with reduced proteolytic activity (e.g., BL21(DE3)pLysS) may improve protein integrity

• Culture Conditions and Induction Parameters:

  • Temperature: Lower induction temperatures (16-25°C) often improve soluble protein yield

  • Induction time: Extended induction periods (overnight) at lower temperatures

  • Inducer concentration: Typically 0.1-0.5 mM IPTG, with lower concentrations favoring soluble expression

  • Media composition: Enriched media (TB, 2xYT) generally provide higher biomass and protein yields

• Purification Strategy:

  • Two-step purification typically yields protein of >85% purity as required for research applications

  • Initial capture via affinity chromatography (Ni-NTA for His-tagged protein)

  • Polishing step using size exclusion or ion exchange chromatography

  • Buffer optimization to maintain stability (typically including 10-20% glycerol and reducing agents)

• Activity Preservation:

  • Addition of stabilizing agents (glycerol, reducing agents)

  • Storage at -20°C for short-term or -80°C for long-term stability

  • Avoiding repeated freeze-thaw cycles

  • Aliquoting purified protein in working volumes

The optimal expression protocol based on available information would involve expression in E. coli BL21(DE3) using a pET-28a vector, induction with 0.2 mM IPTG at 18°C overnight, followed by purification via Ni-NTA chromatography and size exclusion chromatography, with storage in buffer containing 50% glycerol at -80°C for maximum stability .

What are the recommended protocols for assessing inhibitor efficacy against Citrobacter koseri Thymidylate kinase?

A comprehensive inhibitor assessment strategy should include multiple complementary assays:

• Primary Enzymatic Inhibition Assay:

  • Concentration-response testing (IC₅₀ determination)

  • Mode of inhibition characterization (competitive, noncompetitive, uncompetitive)

  • Time-dependency assessment (reversible vs. irreversible inhibition)

  • Recommended method: Coupled spectrophotometric assay with ADP-dependent NADH oxidation

• Binding Affinity Measurements:

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters (ΔH, ΔS, Kd)

  • Surface Plasmon Resonance (SPR) for kinetic binding parameters (kon, koff)

  • Thermal Shift Assay (TSA) for rapid screening and stability assessment

• Selectivity Profiling:

  • Counter-screening against human thymidylate kinase

  • Profiling against related bacterial enzymes

  • Testing against a panel of unrelated kinases to determine specificity

• Cellular Activity Assessment:

  • Antimicrobial activity against C. koseri (MIC determination)

  • Activity against resistant strains

  • Cytotoxicity evaluation against mammalian cell lines

  • Target engagement verification in bacterial cells

• Mechanism of Action Validation:

  • Metabolite profiling to confirm pathway inhibition

  • Resistance development studies

  • Correlation between enzymatic inhibition and cellular activity

A standardized inhibition assay protocol might include:

• Enzyme concentration: 10-50 nM

• Substrate concentrations: At or below Km values (typically 10-50 μM for both dTMP and ATP)

• Buffer: 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 0.1 mg/mL BSA, 1 mM DTT

• Temperature: 30°C

• Preincubation of enzyme with inhibitor: 10-15 minutes

• Data analysis: Nonlinear regression for IC₅₀ determination and enzyme kinetic modeling for mode of inhibition

What techniques can be employed to investigate the catalytic mechanism of Citrobacter koseri Thymidylate kinase?

Elucidating the catalytic mechanism of C. koseri Thymidylate kinase requires a multidisciplinary approach combining structural, biochemical, and computational methods:

• Site-Directed Mutagenesis Studies:

  • Systematic mutation of putative catalytic residues

  • Analysis of kinetic parameters (kcat, Km) for mutants

  • Double-mutant cycle analysis to assess cooperative interactions

  • Active site residues can be identified based on sequence homology with other characterized bacterial thymidylate kinases

• Structural Studies:

  • X-ray crystallography of enzyme in different states (apo, substrate-bound, transition state analog-bound)

  • Cryo-EM analysis for conformational dynamics

  • NMR studies for solution-state dynamics and ligand binding

• Pre-Steady-State Kinetics:

  • Rapid kinetic methods (stopped-flow, quench-flow)

  • Identification and characterization of reaction intermediates

  • Determination of rate-limiting steps

• Isotope Effects and Chemical Modification:

  • Kinetic isotope effects to probe transition state structure

  • Chemical modification of specific residues to assess functional roles

  • Incorporation of non-natural amino acids to probe specific interactions

• Computational Approaches:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations

  • Free energy calculations for the reaction pathway

  • Molecular dynamics simulations of conformational changes

The active site region likely involves residues equivalent to those identified in related thymidylate kinases, particularly the P-loop motif (GXXGXGKT) responsible for nucleotide binding . Based on sequence analysis, the conserved serine residue in position 9 of the active site peptide is likely critical for catalysis, similar to what has been observed in other kinases .

How can Recombinant Citrobacter koseri Thymidylate kinase research contribute to developing new antimicrobial strategies?

Research on C. koseri Thymidylate kinase offers several promising avenues for antimicrobial development:

• Novel Drug Target Validation: Thymidylate kinase represents an underexplored target in the nucleotide synthesis pathway. Establishing its essentiality in C. koseri through genetic approaches and demonstrating that its inhibition leads to bacterial death provides validation for drug development efforts .

• Structure-Based Drug Design: The availability of recombinant C. koseri tmk enables structural studies that can inform rational design of selective inhibitors. These efforts can leverage the subtle differences between bacterial and human thymidylate kinases to achieve selectivity.

• Combination Therapy Approaches: Inhibitors of thymidylate kinase could potentially synergize with existing antibiotics by:

  • Disrupting nucleotide metabolism, weakening bacterial defenses

  • Preventing DNA repair mechanisms, enhancing the efficacy of DNA-damaging antibiotics

  • Reducing mutation rates that lead to resistance development

• Alternatives to Traditional Antibiotics: Instead of directly killing bacteria, tmk inhibitors might be developed as anti-virulence agents that reduce pathogenicity or as sensitizing agents that make resistant strains susceptible to conventional antibiotics again.

• Broad-Spectrum Potential: The conservation of thymidylate kinase across bacterial species suggests that inhibitors might show activity against multiple pathogens, including other problematic Enterobacteriaceae with antibiotic resistance.

The increasing antibiotic resistance in C. koseri strains makes this research particularly timely and significant . By targeting an essential enzyme in a critical pathway using structure-based approaches, researchers can develop antimicrobials with novel mechanisms of action, potentially overcoming existing resistance mechanisms.

What role does Citrobacter koseri Thymidylate kinase play in bacterial pathogenesis and antimicrobial resistance?

Thymidylate kinase's role in C. koseri pathogenesis and antimicrobial resistance is multifaceted:

• Essential Metabolic Function: As a critical enzyme in the thymidine nucleotide synthesis pathway, tmk is essential for DNA replication and bacterial survival. Its inhibition would prevent bacterial proliferation during infection .

• Stress Response and Adaptation: During infection and antibiotic exposure, bacteria often upregulate nucleotide metabolism pathways to:

  • Support increased DNA repair needed under stress conditions

  • Maintain DNA replication fidelity under challenging conditions

  • Provide metabolic precursors for stress response mechanisms

• Relationship to Antimicrobial Resistance:

  • Mutations in tmk genes can potentially affect susceptibility to certain antimicrobials, especially those targeting nucleic acid metabolism

  • Altered expression levels of tmk may contribute to fitness compensation in resistant strains

  • Nucleotide pool imbalances caused by antibiotic pressure may be compensated by changes in tmk activity

• Biofilm Formation: Nucleotide metabolism has been implicated in biofilm formation, a key virulence factor for C. koseri that can contribute to persistent infections and treatment failures. Thymidylate kinase may indirectly influence biofilm development through its effects on nucleotide pools.

• Host-Pathogen Interactions: During infection, bacterial nucleotide metabolism must adapt to the host environment where nutrient availability differs significantly from laboratory conditions. Thymidylate kinase activity may be modulated during this adaptation process.

Research suggests that C. koseri has acquired resistance to multiple conventional antibiotics , highlighting the need for novel targets like tmk. Targeting this enzyme represents a promising strategy to overcome existing resistance mechanisms by exploiting a previously untargeted essential pathway.

What future research directions hold the most promise for Citrobacter koseri Thymidylate kinase studies?

Several high-potential research directions for C. koseri Thymidylate kinase include:

• Structural Biology and Dynamics:

  • Obtaining high-resolution crystal structures of C. koseri tmk in multiple conformational states

  • Utilizing cryo-EM to capture conformational ensembles

  • Employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamics

  • Developing computational models to predict conformational changes during catalysis

• Inhibitor Development:

  • Fragment-based screening approaches to identify novel chemical scaffolds

  • Structure-based design of transition state analogs

  • Development of covalent inhibitors targeting non-conserved cysteine residues

  • Allosteric inhibitor discovery focusing on regions distant from the active site

• Systems Biology Integration:

  • Investigating metabolic network effects of tmk inhibition

  • Exploring synthetic lethality with other targets

  • Metabolomic profiling to understand downstream effects of tmk inhibition

  • Integration with multi-omics approaches to elucidate resistance mechanisms

• Translational Research:

  • Development of cell-penetrant inhibitors effective against C. koseri

  • In vivo validation in infection models

  • Combination studies with existing antibiotics

  • PK/PD modeling to optimize dosing strategies for tmk inhibitors

• Genetic and Physiological Studies:

  • CRISPR interference studies to validate essentiality under different conditions

  • Conditional knockdown systems to study tmk function in vivo

  • Investigation of natural variation in tmk sequences across clinical isolates

  • Relationship between tmk expression and virulence in animal models

The development of multi-epitope vaccines targeting C. koseri, as mentioned in the research literature , represents a complementary approach that could be explored alongside tmk inhibitor development. An integrated strategy combining both enzyme inhibition and immunological approaches might provide the most robust defense against this increasingly resistant pathogen.