Recombinant Citrobacter koseri Argininosuccinate synthase (argG)

What is Citrobacter koseri argininosuccinate synthase and what is its role in bacterial metabolism?

Argininosuccinate synthase (encoded by the argG gene) is a critical enzyme in the arginine biosynthetic pathway of Citrobacter koseri. It catalyzes the ATP-dependent condensation of citrulline and aspartate to form argininosuccinate, the immediate precursor of arginine. In C. koseri, this enzyme plays an essential role in nitrogen metabolism and amino acid biosynthesis. The enzyme functions within a metabolic network where arginine serves not only as a protein building block but also as a precursor for various cellular processes . Unlike in some other bacterial species, C. koseri's argG appears to have specific structural features that may contribute to the organism's pathogenicity profile and metabolic adaptability in different host environments .

How does C. koseri argG differ from argininosuccinate synthase in other bacterial species?

These differences may contribute to C. koseri's distinctive pathogenic potential, particularly in immunocompromised hosts and neonates .

What are the typical yields and solubility challenges when expressing recombinant C. koseri argG?

Recombinant expression of C. koseri argG presents several challenges typical of bacterial metabolic enzymes. Based on research experience with similar enzymes, the following considerations are important:

Expression yields typically range from 15-30 mg/L in E. coli expression systems when using optimized conditions. Solubility can be a significant challenge due to the enzyme's tendency to form inclusion bodies, particularly at high expression levels. This can be addressed through several strategies:

• Lowering induction temperature to 16-18°C

• Using weaker promoters or lower IPTG concentrations (0.1-0.3 mM)

• Co-expression with chaperones like GroEL/GroES

• Fusion with solubility-enhancing tags such as MBP or SUMO

A typical optimization approach involves testing multiple expression conditions:

The protein typically exhibits good stability when stored in Tris-based buffers with 50% glycerol at -20°C or -80°C, similar to other recombinant proteins from this organism .

What expression system is optimal for high-yield production of functional C. koseri argG?

The optimal expression system for C. koseri argG depends on the specific research objectives. For structural and biochemical studies requiring high yields of pure protein, E. coli-based systems remain the standard choice. The methodological approach should include:

E. coli BL21(DE3) or its derivatives are generally preferred host strains due to their reduced protease activity and efficient transcription machinery. For expression vectors, pET systems with T7 promoters typically provide good control over expression. When expressing C. koseri proteins, codon optimization should be considered as there may be codon usage bias differences between C. koseri and E. coli .

For challenging expression cases, specialized E. coli strains such as Rosetta (for rare codon supplementation) or SHuffle (for disulfide bond formation) may be employed. The use of fusion tags can dramatically impact both yield and solubility:

A systematic comparison of expression conditions is recommended, with optimal results typically achieved with BL21(DE3) hosts, using TB media, induction at OD600 of 0.6-0.8 with 0.2-0.3 mM IPTG, and expression at 18°C for 16-20 hours .

What purification strategy yields the highest purity and activity retention for recombinant C. koseri argG?

A multi-step purification strategy is typically required to obtain high-purity, active C. koseri argG. Based on biochemical principles and experience with similar enzymes, the following approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged protein

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose at pH 8.0)

• Polishing: Size exclusion chromatography (Superdex 200) to separate oligomeric states and remove aggregates

Critical buffer considerations include:

Activity retention is maximized by maintaining reducing conditions throughout purification, minimizing freeze-thaw cycles, and avoiding prolonged exposure to room temperature. Enzyme activity should be monitored after each purification step to ensure the protocol is not compromising function. Typical final purity should exceed 95% as determined by SDS-PAGE .

How can one establish a reliable activity assay for recombinant C. koseri argG?

Establishing a reliable activity assay for argininosuccinate synthase is essential for both characterization studies and quality control during purification. The following methodological approach is recommended:

The standard assay for argG activity measures the ATP-dependent conversion of citrulline and aspartate to argininosuccinate. Two primary detection methods are commonly employed:

• Coupled enzyme assay: This monitors AMP production (a byproduct of the reaction) by coupling to additional enzymes (adenylate kinase and pyruvate kinase/lactate dehydrogenase) and following NADH oxidation spectrophotometrically at 340 nm.

• Direct product detection: Using HPLC or LC-MS to quantify argininosuccinate formation directly.

Assay conditions optimization should include:

Control experiments should include enzyme-minus, substrate-minus, and heat-inactivated enzyme controls. For kinetic analysis, substrate concentrations should be varied systematically while maintaining other components at saturation to determine KM and Vmax values. When comparing wild-type and mutant versions of the enzyme, or enzyme preparations from different purification batches, standardized specific activity measurements (μmol product/min/mg protein) should be used .

What structural features distinguish C. koseri argG from other bacterial argininosuccinate synthases?

Although complete structural data for C. koseri argG is limited in the provided search results, comparative analysis with homologous enzymes suggests several distinguishing features:

The catalytic core of argininosuccinate synthase is generally conserved across species, with distinctive differences appearing primarily in substrate-binding regions and surface-exposed loops. In C. koseri, these regions may have evolved specific adaptations related to its pathogenic lifestyle.

Computational structure-based analysis methods, similar to those described for enzyme redesign, can help identify unique features of C. koseri argG . Key structural features likely include:

• Nucleotide-binding domain for ATP coordination

• Specific binding pockets for citrulline and aspartate

• Interfacial regions that facilitate oligomerization (typically tetrameric in bacterial argGs)

• Surface features that may interact with other cellular components

The sequence of C. koseri proteins can vary significantly from other bacterial species, even within the Citrobacter genus, as demonstrated by phylogenetic studies showing distinct clustering patterns . These sequence variations likely translate to structural differences that could affect substrate specificity, catalytic efficiency, and regulatory mechanisms.

To fully characterize these differences, techniques including X-ray crystallography, cryo-EM, or computational modeling based on homologous structures would be required. Molecular dynamics simulations can further reveal dynamic aspects of substrate binding and catalysis that distinguish the C. koseri enzyme from its homologs .

How does site-directed mutagenesis inform the functional understanding of C. koseri argG?

Site-directed mutagenesis represents a powerful approach to probe structure-function relationships in C. koseri argG. Building on computational structure-based redesign principles , a systematic mutagenesis strategy should target:

• Catalytic residues: Mutations in the active site residues directly involved in catalysis typically abolish activity completely. These experiments confirm the essential catalytic mechanism.

• Substrate-binding residues: Mutations affecting substrate binding often alter KM values without dramatically changing kcat, providing insights into substrate specificity.

• Allosteric sites: Mutations at potential regulatory sites may affect cooperativity or response to allosteric effectors.

• Interface residues: Mutations at oligomerization interfaces can disrupt quaternary structure and often diminish activity.

A methodical approach to mutation design includes:

Analysis of mutant enzymes should include full kinetic characterization (kcat, KM for all substrates), stability assessment (thermal denaturation, proteolytic susceptibility), and when possible, structural characterization (to confirm the mutation did not cause gross structural changes).

The integration of computational prediction with experimental validation, as demonstrated in enzyme redesign studies , provides particularly powerful insights into the functional significance of specific residues and structural features.

What are the primary challenges in crystallizing C. koseri argG for structural studies?

Crystallizing C. koseri argG for structural studies presents several challenges that require methodical approaches to overcome:

• Protein purity and homogeneity: Argininosuccinate synthase must be purified to >95% purity with minimal heterogeneity. Size-exclusion chromatography as a final purification step is essential to isolate a monodisperse population.

• Stability during concentration: The enzyme may show aggregation tendencies during concentration to levels required for crystallization (typically 5-15 mg/ml). This can be addressed by:

  • Optimizing buffer conditions (pH, salt concentration)

  • Including stabilizing additives (glycerol, reducing agents)

  • Using gentle concentration methods

• Conformational flexibility: Like many enzymes, argG likely exhibits conformational dynamics that can impede crystallization. Strategies to address this include:

  • Co-crystallization with substrates, products, or non-hydrolyzable substrate analogs

  • Use of ligands that lock the enzyme in specific conformations

  • Limited proteolysis to remove flexible regions

• Crystal optimization: Initial crystals often diffract poorly and require optimization. A systematic grid screen approach is recommended:

Alternative approaches when traditional crystallization fails include:

• Crystallization of individual domains

• Surface entropy reduction mutagenesis

• Fusion with crystallization chaperones

• Cryo-EM as an alternative structural determination method, especially valuable for larger complexes

Successful crystallization will likely require iterative optimization based on initial screening results, with careful attention to protein batch consistency between trials .

How does argG contribute to the pathogenicity of Citrobacter koseri in immunocompromised hosts?

Citrobacter koseri is known to cause opportunistic infections, particularly in neonates and immunocompromised individuals, with severe manifestations including meningitis, brain abscesses, and epidural spinal abscesses . While argG itself has not been directly implicated as a virulence factor in the provided search results, several hypotheses can be formulated regarding its potential contribution to pathogenicity:

• Metabolic adaptation: ArgG functions in arginine biosynthesis, which may be critical for bacterial survival in host microenvironments where this amino acid is limited. This could provide a metabolic advantage during infection.

• Host immune interaction: Bacterial proteins, including metabolic enzymes, can sometimes serve dual roles and interact with host immune components. Recent research has identified C. koseri proteins with antigenic potential that could be vaccine targets .

• Stress response: In pathogenic bacteria, metabolic enzymes often play roles in stress adaptation, which may contribute to survival in hostile host environments. The ability to maintain arginine biosynthesis under stress conditions could enhance persistence.

• Relationship to iron acquisition: The search results mention that C. koseri contains a high-pathogenicity island (HPI) cluster related to iron transport that contributes significantly to its virulence . While not directly connected to argG in the provided information, metabolic pathways and iron acquisition systems often have regulatory interconnections.

Research approaches to investigate argG's role in pathogenicity should include:

• Construction of argG knockout mutants to assess virulence in animal models

• Transcriptomic analysis of argG expression during infection

• Evaluation of argG expression under various stress conditions relevant to host environments

• Investigation of potential moonlighting functions beyond its primary metabolic role

What experimental approaches best demonstrate the potential of C. koseri argG as a drug target?

Establishing C. koseri argG as a viable drug target requires a systematic experimental approach to validate its essentiality, druggability, and specificity:

• Target validation should begin with genetic approaches:

  • Gene knockout studies using CRISPR-Cas9 or traditional methods to determine if argG is essential for C. koseri growth, particularly under conditions mimicking the host environment

  • Conditional knockdown systems (e.g., inducible antisense RNA) to demonstrate that reduced argG levels compromise bacterial viability

  • Complementation studies to confirm phenotypes are specifically due to argG disruption

• Biochemical characterization to assess druggability:

  • High-resolution structural analysis of the enzyme's active site to identify potential binding pockets

  • Development of high-throughput activity assays suitable for inhibitor screening

  • Initial screening with known inhibitors of homologous enzymes to establish proof-of-concept

• Differential targeting potential:

  • Comparative analysis of C. koseri argG with human argininosuccinate synthase to identify structural differences that could be exploited for selective inhibition

  • Modeling studies to predict selective binding to bacterial versus human enzyme

• Preliminary inhibitor development:

  • Structure-based design of potential inhibitors targeting unique features of C. koseri argG

  • Fragment-based screening approaches to identify initial binding molecules

  • Biochemical validation of binding and inhibition using purified recombinant enzyme

• Cellular validation:

  • Testing candidate inhibitors against C. koseri cultures to demonstrate growth inhibition

  • Metabolomic analysis to confirm that inhibition specifically affects the arginine biosynthesis pathway

  • Cytotoxicity testing against mammalian cells to establish a preliminary therapeutic window

The computational structure-based approach described for enzyme redesign could be adapted for inhibitor design, potentially identifying molecules that selectively interact with unique features of the C. koseri enzyme .

How can recombinant C. koseri argG be utilized in vaccine development research?

Recent research has identified C. koseri proteins with antigenic potential as possible vaccine targets . While argG is not explicitly mentioned as a primary vaccine candidate in the search results, recombinant argG could be utilized in vaccine development research through several approaches:

• Antigenicity assessment: Recombinant argG can be evaluated for its ability to stimulate immune responses:

  • In silico epitope prediction to identify potential B-cell and T-cell epitopes

  • ELISA-based antibody binding assays using sera from patients recovered from C. koseri infections

  • T-cell activation assays to assess cell-mediated immune responses

• Subunit vaccine development: If argG demonstrates sufficient antigenicity, it could be explored as a component of a subunit vaccine:

  • Expression and purification of immunodominant domains

  • Conjugation to carrier proteins to enhance immunogenicity

  • Formulation with appropriate adjuvants to direct the desired immune response

• Reverse vaccinology approach: The subtractive proteomics methodology mentioned for C. koseri represents a systematic approach to identify promising vaccine candidates:

  • Computational prediction of surface-exposed or secreted proteins

  • Analysis of conservation across C. koseri strains

  • Exclusion of proteins with human homologs to minimize autoimmunity risk

  • Recombinant expression and immunological testing of candidate antigens

• Vaccine efficacy testing:

  • Development of animal models of C. koseri infection

  • Immunization protocols to evaluate protective efficacy

  • Challenge studies to assess protection against infection

• Multi-antigen approaches:

  • Combination of argG with other identified antigens to create a multi-component vaccine

  • Evaluation of synergistic immune responses

This approach aligns with the emerging vaccine development strategy identified in the search results, which emphasizes the importance of identifying antigenic proteins to design effective vaccines against C. koseri, particularly given the increasing antibiotic resistance observed in this pathogen .

How can computational modeling enhance our understanding of C. koseri argG catalytic mechanism?

Computational modeling offers powerful insights into the catalytic mechanism of C. koseri argG, building upon approaches similar to those described for enzyme redesign :

• Homology modeling: When experimental structures are unavailable, homology modeling based on related bacterial argininosuccinate synthases provides a starting point for mechanistic studies:

  • Template selection based on sequence similarity and functional conservation

  • Model refinement using energy minimization and molecular dynamics

  • Validation through comparison with biochemical data

• Molecular dynamics (MD) simulations: MD can reveal dynamic aspects of enzyme function:

  • Conformational changes associated with substrate binding

  • Water molecule movements in the active site

  • Identification of transient binding pockets not visible in static structures

  • Allosteric communication networks within the protein

• Quantum mechanics/molecular mechanics (QM/MM) calculations: For detailed understanding of bond formation/breaking:

  • Hybrid calculations with QM treatment of the active site and MM for the rest of the protein

  • Energy profiles for the reaction coordinate

  • Identification of transition states and intermediates

  • Evaluation of alternative reaction mechanisms

• Structure-based predictions:

  • Identification of critical catalytic residues

  • Prediction of the effects of mutations on activity

  • Virtual screening for potential inhibitors

These computational approaches generate testable hypotheses that can guide experimental work, such as site-directed mutagenesis of predicted catalytic residues or the design of transition-state analogs as potential inhibitors. The computational structure-based redesign methodology described in the search results provides a framework that could be adapted specifically for mechanistic studies of C. koseri argG.

What approaches can resolve contradictory findings in C. koseri argG kinetic studies?

Resolving contradictory findings in enzyme kinetic studies requires a systematic approach to identify and address sources of variability:

• Standardization of enzyme preparation:

  • Establish consistent expression and purification protocols

  • Characterize each preparation for purity, specific activity, and oligomeric state

  • Document batch-to-batch variation and establish acceptance criteria

• Assay methodology validation:

  • Compare different assay methods (e.g., coupled enzyme assay vs. direct product detection)

  • Validate linearity, sensitivity, and specificity of each assay

  • Establish standard curves and determine lower limits of detection

• Experimental design to identify variables affecting kinetics:

  • Systematic variation of buffer components (pH, salt concentration, divalent cations)

  • Temperature dependence studies

  • Evaluation of potential activators or inhibitors present in different preparations

• Statistical approaches to resolve discrepancies:

  • Meta-analysis of multiple independent studies

  • Bayesian analysis to incorporate prior knowledge

  • Sensitivity analysis to identify parameters with greatest impact on results

• Advanced kinetic modeling:

  • Global fitting of multiple datasets

  • Testing of alternative kinetic models beyond basic Michaelis-Menten

  • Incorporation of enzyme conformational changes into kinetic models

When facing contradictory results, it is essential to avoid confirmation bias and to systematically evaluate all potential sources of variation. Publication of comprehensive methods, including detailed buffer compositions and enzyme preparation protocols, is crucial for reproducibility across laboratories .

How can isotope labeling and NMR spectroscopy provide insights into C. koseri argG reaction mechanism?

Isotope labeling combined with NMR spectroscopy offers powerful approaches to elucidate the detailed reaction mechanism of C. koseri argininosuccinate synthase:

• Substrate tracking using isotope-labeled precursors:

  • ¹³C-labeled citrulline and aspartate can track carbon incorporation into argininosuccinate

  • ¹⁵N-labeled substrates can track nitrogen transfer

  • ¹⁸O-labeled water or substrates can identify oxygen incorporation or exchange

• Reaction intermediate identification:

  • Rapid quench techniques coupled with NMR analysis to trap transient intermediates

  • Temperature variation to slow reaction kinetics and capture intermediates

  • Use of substrate analogs that form stable complexes at specific reaction steps

• Enzyme-substrate interactions:

  • HSQC (Heteronuclear Single Quantum Coherence) experiments with ¹⁵N-labeled enzyme to monitor chemical shift perturbations upon substrate binding

  • Saturation transfer difference (STD) NMR to map substrate binding epitopes

  • Transfer NOE experiments to determine bound substrate conformation

• Dynamic aspects of catalysis:

  • ¹⁵N relaxation measurements to characterize backbone dynamics

  • Hydrogen/deuterium exchange to identify regions with altered solvent accessibility during catalysis

  • CPMG relaxation dispersion to detect conformational exchange processes

• Methodological considerations:

  • Enzyme concentration requirements (typically 0.1-1 mM)

  • Time stability of samples during lengthy NMR experiments

  • Potential need for deuterated enzyme to improve spectral quality

The integration of NMR data with computational modeling (as described in 5.1) can provide a comprehensive understanding of the reaction mechanism at atomic resolution. This approach is particularly valuable for distinguishing between alternative mechanistic hypotheses and for identifying transient intermediates that may be difficult to detect by other means .

How does the genetic diversity of argG across Citrobacter species reflect evolutionary adaptations?

The genetic diversity of argG across Citrobacter species provides important insights into evolutionary adaptations of these bacteria. Based on the genomic analyses described in the search results:

Citrobacter encompasses at least 11 distinct species or genomic groups, with C. koseri forming a distinct cluster in phylogenetic analyses based on whole genome sequencing (WGS) data . This genomic differentiation likely extends to metabolic genes like argG. Comparative analysis of argG sequences across these groups can reveal:

• Evolutionary patterns:

  • Core vs. accessory gene status of argG in Citrobacter species

  • Evidence of horizontal gene transfer vs. vertical inheritance

  • Selection pressures acting on different domains of the protein

• Functional adaptations:

  • Correlation between argG sequence variations and ecological niches

  • Adaptive changes in pathogenic vs. non-pathogenic Citrobacter species

  • Relationship between argG variants and host specificity

The distinct clustering of C. koseri strains observed in whole-genome phylogenetic analyses suggests that their metabolic genes, including argG, may have undergone specific evolutionary adaptations related to their pathogenic potential, particularly in meningitis and brain abscess formation in neonates and immunocompromised individuals.

Analysis methods should include:

• Calculation of dN/dS ratios to identify regions under positive or purifying selection

• Bayesian evolutionary analysis to reconstruct ancestral sequences

• Correlation of sequence changes with known functional differences

• Structural mapping of variable residues to identify potential functional significance

Understanding these evolutionary patterns can provide insights into C. koseri's specific adaptations and virulence mechanisms, potentially identifying targets for therapeutic intervention .

What comparative genomic approaches can identify functional partners of C. koseri argG?

Comparative genomic approaches offer powerful methods to identify functional partners and regulatory networks associated with C. koseri argG:

• Gene neighborhood analysis:

  • Examination of genomic context around argG across Citrobacter species

  • Identification of conserved gene clusters suggesting functional relationships

  • Comparison with other Enterobacteriaceae to identify genus-specific arrangements

• Phylogenetic profiling:

  • Correlation of argG presence/absence patterns with other genes across bacterial species

  • Identification of co-evolving gene pairs suggesting functional relationships

  • Clustering of genes with similar phylogenetic profiles

• Gene expression correlation:

  • Analysis of transcriptomic data to identify genes co-regulated with argG

  • Identification of condition-specific co-expression patterns

  • Network analysis to place argG in broader regulatory frameworks

• Protein-protein interaction prediction:

  • Computational prediction of physical interactions based on sequence/structure

  • Identification of conserved protein domains suggesting interaction potential

  • Comparison with experimentally validated interactions in related species

The whole-genome sequence (WGS) data available for multiple Citrobacter isolates provides an excellent foundation for these comparative approaches. The systematic comparative genomic analyses that have been performed for virulence factors, resistance genes, and macromolecular secretion systems among Citrobacter species could be extended specifically to argG and its functional partners.

How can recombinant C. koseri argG be used to study metabolic adaptation during infection?

Recombinant C. koseri argG serves as a valuable tool to investigate metabolic adaptations during infection processes:

• In vitro modeling of host conditions:

  • Biochemical characterization of argG activity under conditions mimicking different host environments (pH, nutrient availability, oxidative stress)

  • Determination of kinetic parameters under physiologically relevant conditions

  • Comparison with homologous enzymes from non-pathogenic bacteria

• Structural adaptations to host environments:

  • Stability analysis under conditions encountered during infection

  • Identification of structural features that confer resistance to host defense mechanisms

  • Comparison of substrate specificity with host enzyme counterparts

• Interaction studies with host factors:

  • Binding studies with host proteins or metabolites

  • Investigation of potential inhibition by host-derived molecules

  • Identification of post-translational modifications induced by host factors

• Systems biology approaches:

  • Integration of argG activity data into metabolic flux models

  • Prediction of metabolic bottlenecks during infection

  • Simulation of metabolic responses to changing host environments

• Translation to in vivo studies:

  • Development of reporter systems to monitor argG activity in vivo

  • Creation of argG variants with altered regulatory properties

  • Testing hypotheses generated from in vitro studies in animal infection models

This research is particularly relevant given C. koseri's role in serious infections, especially in neonates and immunocompromised individuals . The identification of key gene clusters in C. koseri that are absent in other Citrobacter species, such as the high-pathogenicity island (HPI) cluster for iron transport , suggests that metabolic adaptations play a crucial role in its pathogenicity. Similar specialized adaptations may exist in arginine metabolism pathways that could be revealed through detailed study of recombinant argG.