Recombinant Protochlamydia amoebophila Guanylate kinase (gmk)

What is Protochlamydia amoebophila and why is its guanylate kinase significant to study?

Protochlamydia amoebophila UWE25 is an obligate intracellular symbiont belonging to the Chlamydiales order that thrives within the protozoan host Acanthamoeba sp . Unlike its pathogenic relatives in the Chlamydiaceae family (which cause human diseases), P. amoebophila represents an evolutionary important organism for understanding chlamydial biology.

The guanylate kinase (gmk) from P. amoebophila is significant for several reasons:

• It plays an essential role in nucleotide metabolism, catalyzing the phosphorylation of GMP to GDP, which serves as a precursor for GTP synthesis

• P. amoebophila lacks complete de novo nucleotide synthesis pathways, making nucleotide metabolism enzymes critical for survival

• Studying gmk provides insights into the metabolic adaptation of obligate intracellular bacteria to their host environments

• Comparative analysis with gmk from pathogenic chlamydiae can reveal evolutionary adaptations and potential antimicrobial targets

How does guanylate kinase function within the metabolic network of P. amoebophila?

Guanylate kinase functions within a critically important pathway in P. amoebophila's metabolic network:

• P. amoebophila cannot synthesize purine nucleotides de novo due to genomic reduction associated with its intracellular lifestyle

• The organism depends on host-derived GMP, which is phosphorylated by gmk to produce GDP

• GDP is subsequently converted to GTP by nucleoside-diphosphate kinase (ndk), also encoded in the P. amoebophila genome

• GTP serves multiple essential functions:

  • Energy source for protein synthesis

  • Substrate for RNA synthesis

  • Precursor for dGTP used in DNA synthesis

  • Signaling molecule

This metabolic framework explains why gmk is an essential enzyme for P. amoebophila survival. The pathways are further integrated with the host cell metabolism through specialized nucleotide transport proteins, particularly the nucleotide transporter (NTT) family members that import nucleotides from the host .

What expression systems are most effective for recombinant P. amoebophila gmk production?

Based on methodologies used for similar proteins, E. coli expression systems are most effective for recombinant P. amoebophila gmk production. The experimental procedure typically follows:

• Gene amplification from P. amoebophila genomic DNA using PCR with primers containing appropriate restriction sites (e.g., XhoI and BamHI)

• Cloning into an expression vector such as pET16b (Novagen) which provides:

  • N-terminal His-tag for purification

  • T7 promoter for high-level expression

  • Ampicillin resistance marker for selection

• Transformation into an E. coli expression strain:

  • BL21(DE3) for standard expression

  • Rosetta or Origami strains if codon usage or disulfide bond formation is problematic

Expression optimization typically requires testing multiple conditions:

• Temperature (16°C, 25°C, 37°C)

• IPTG concentration (0.1-1.0 mM)

• Duration of induction (4-16 hours)

• Media composition (LB, TB, or minimal media with supplements)

What is the optimal purification strategy for obtaining active recombinant P. amoebophila gmk?

For optimal purification of enzymatically active recombinant P. amoebophila gmk, the following strategy has proven effective for similar enzymes:

• Cell lysis:

  • Resuspend cells in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole

  • Add protease inhibitors (e.g., PMSF or commercial cocktail)

  • Lyse using sonication or French press

  • Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Immobilized metal affinity chromatography (IMAC):

  • Load clarified lysate onto Ni-NTA column

  • Wash with buffer containing 20-30 mM imidazole

  • Elute with buffer containing 250-300 mM imidazole

• Size exclusion chromatography:

  • Further purify using Superdex 75 or 200 column

  • Use buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂

  • Analyze fractions by SDS-PAGE

• Quality control:

  • Assess purity by SDS-PAGE (>95% purity recommended)

  • Verify identity by Western blot or mass spectrometry

  • Confirm activity using enzyme assays (see section 3.1)

  • Determine protein concentration using Bradford assay or A₂₈₀ measurement

This protocol typically yields 5-10 mg of pure, active enzyme per liter of bacterial culture.

What methods are most reliable for determining the kinetic parameters of recombinant P. amoebophila gmk?

For accurate determination of kinetic parameters of recombinant P. amoebophila gmk, several complementary approaches can be employed:

• Coupled enzyme assay:

  • Measure ADP production using pyruvate kinase and lactate dehydrogenase

  • Monitor NADH oxidation at 340 nm

  • Reaction mixture: 50 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 2 units pyruvate kinase, 2 units lactate dehydrogenase, varying GMP concentrations (5-500 μM), fixed ATP (4 mM), and purified gmk

• Direct measurement of product formation by HPLC:

  • Separate nucleotides on reverse-phase or ion-exchange column

  • Monitor at 260 nm

  • Quantify GDP formation over time at different substrate concentrations

• Radiochemical assay:

  • Use [α-³²P]-GMP as substrate

  • Separate products by thin-layer chromatography

  • Quantify radioactive products using phosphorimager

For data analysis:

• Plot initial velocity versus substrate concentration

• Fit data to Michaelis-Menten equation to determine Km and Vmax

• Calculate kcat by dividing Vmax by enzyme concentration

• For inhibition studies, determine Ki using appropriate inhibition models

Based on data from similar bacterial gmk enzymes, expected kinetic parameters might fall within these ranges :

• kcat: 20-70 sec⁻¹

• Km (GMP): 20-150 μM

• Km (ATP): 100-500 μM

How does (p)ppGpp regulate P. amoebophila gmk activity and what are the structural determinants?

While specific data on P. amoebophila gmk regulation by (p)ppGpp is not directly available, comparative analysis with other bacterial gmk enzymes provides valuable insights:

(p)ppGpp is a bacterial alarmone that mediates stress responses, and its interaction with gmk represents an important regulatory mechanism. In many bacteria, (p)ppGpp competitively inhibits gmk activity, preventing GMP conversion to GDP and subsequently limiting GTP synthesis during stress conditions .

The structural determinants for (p)ppGpp binding likely include:

• Binding pocket residues: Conserved residues in the GMP binding site, particularly a tyrosine residue (equivalent to Tyr78 in S. aureus gmk) that contributes significantly to (p)ppGpp binding affinity

• Mode of inhibition: Competitive inhibition with respect to GMP, indicating that (p)ppGpp binds to the GMP binding site

• Phylogenetic patterns: The sensitivity to (p)ppGpp varies across bacterial phyla:

  • Firmicutes, Actinobacteria, and Deinococcus-Thermus show strong (p)ppGpp-gmk interaction

  • Proteobacteria typically show resistance to (p)ppGpp regulation at the gmk level

Since P. amoebophila is evolutionarily distinct from the well-characterized bacterial groups, determining its gmk sensitivity to (p)ppGpp would provide valuable information about the conservation of this regulatory mechanism across bacterial lineages.

Based on patterns observed in other bacteria, the inhibition constant (Ki) for P. amoebophila gmk could range from 1-60 μM for pppGpp, with potential variation between ppGpp and pppGpp .

What is the impact of pH, temperature, and divalent cations on P. amoebophila gmk activity?

For optimal characterization of P. amoebophila gmk activity, the following environmental parameters should be systematically evaluated:

• pH dependence:

  • Test activity across pH range 6.0-9.0 using appropriate buffers

  • Most bacterial gmk enzymes show optimal activity at pH 7.0-8.0

  • Use overlapping buffers (MES, HEPES, Tris) to eliminate buffer-specific effects

• Temperature effects:

  • Determine temperature optimum by assaying at 10-60°C

  • Assess thermal stability by pre-incubating enzyme at various temperatures before assaying

  • P. amoebophila grows in Acanthamoeba at temperatures around 20-30°C, suggesting its enzymes may be adapted to this range

• Divalent cation requirements:

  • Gmk requires divalent cations for activity, typically Mg²⁺

  • Test various concentrations (1-20 mM) of Mg²⁺, Mn²⁺, Ca²⁺, and other divalent cations

  • Determine if monovalent cations (K⁺, Na⁺) affect activity

• Stability conditions:

  • Evaluate enzyme stability in different storage buffers

  • Test effects of additives (glycerol, DTT, BSA) on long-term stability

  • Determine freeze-thaw stability

These parameters are critical for establishing reproducible assay conditions and for comparing P. amoebophila gmk properties with those of other bacterial gmk enzymes.

How does P. amoebophila gmk differ from gmk enzymes in pathogenic Chlamydiaceae?

P. amoebophila gmk likely exhibits several differences from gmk enzymes in pathogenic Chlamydiaceae, reflecting their evolutionary divergence and adaptation to different host environments:

These differences could be exploited for developing selective inhibitors targeting pathogenic Chlamydiaceae without affecting related environmental species.

What does phylogenetic analysis of gmk reveal about the evolution of nucleotide metabolism in Chlamydiales?

Phylogenetic analysis of gmk provides valuable insights into the evolution of nucleotide metabolism in Chlamydiales:

• Conservation of essential function:

  • Gmk is conserved across all Chlamydiales despite genomic reduction during adaptation to intracellular lifestyle

  • This conservation highlights the essential nature of GMP->GDP conversion in these organisms

• Adaptation to intracellular niche:

  • Chlamydiales have lost de novo nucleotide synthesis pathways but retained key enzymes like gmk for nucleotide interconversion

  • This pattern reflects a metabolic strategy focused on scavenging host nucleotides rather than de novo synthesis

• Co-evolution with nucleotide transporters:

  • The presence of specialized nucleotide transporters (NTTs) in Chlamydiales correlates with the retention of gmk

  • P. amoebophila's five NTT transporters with distinct substrate specificities suggest a sophisticated system for nucleotide acquisition that works in concert with gmk

• Regulatory diversification:

  • Variations in gmk regulation across Chlamydiales lineages may reflect adaptation to different host environments

  • The (p)ppGpp regulatory system shows phylogenetic patterns that might extend to Chlamydiales

• Horizontal gene transfer assessment:

  • Analysis of gmk sequences can help identify potential horizontal gene transfer events in the evolution of Chlamydiales

  • Unusual sequence features or unexpected phylogenetic placement would suggest horizontal acquisition

This evolutionary analysis provides context for understanding P. amoebophila gmk function and its role in the adaptation to an intracellular lifestyle.

How can site-directed mutagenesis of recombinant P. amoebophila gmk inform structure-function relationships?

Site-directed mutagenesis of recombinant P. amoebophila gmk can provide significant insights into structure-function relationships through strategic amino acid substitutions:

• Catalytic site mutations:

  • Identify conserved residues involved in GMP binding by sequence alignment with characterized gmk enzymes

  • Create alanine substitutions of putative catalytic residues

  • Measure effects on Km and kcat to determine contribution to substrate binding and catalysis

• (p)ppGpp sensitivity determinants:

  • Target residues equivalent to Tyr78 in S. aureus gmk, which significantly affects (p)ppGpp binding

  • Create variants mimicking the Y78F mutation that reduced (p)ppGpp sensitivity in B. subtilis gmk

  • Measure inhibition constants for (p)ppGpp to identify key regulatory residues

• Domain interface mutations:

  • If P. amoebophila gmk functions as an oligomer, mutate residues at subunit interfaces

  • Analyze effects on oligomerization and cooperative kinetics

  • Correlate structural changes with functional alterations

• Substrate specificity engineering:

  • Design mutations to alter nucleotide specificity (e.g., modify GMP binding site to accept other nucleotides)

  • Test activity with non-canonical substrates

  • Evaluate evolutionary constraints on substrate specificity

A methodical mutagenesis strategy might include:

• Sequence alignment to identify conserved and divergent residues

• Homology modeling to predict structural impacts of mutations

• Creation of single and multiple mutations using PCR-based methods

• Parallel purification and characterization of multiple variants

• Circular dichroism to verify proper folding of variants

• Detailed kinetic analysis of each variant

What are effective approaches for screening potential inhibitors of P. amoebophila gmk?

For effective screening of potential P. amoebophila gmk inhibitors, a comprehensive strategy incorporating multiple approaches is recommended:

• High-throughput primary screening:

  • Adapt the coupled enzyme assay to 96 or 384-well format

  • Screen diverse chemical libraries at single concentration (10-50 μM)

  • Define hit criteria (e.g., >50% inhibition)

  • Include positive controls (known nucleotide analogs) and negative controls

• Dose-response confirmation:

  • Test hits in dose-response format (0.1-100 μM)

  • Determine IC₅₀ values and Hill coefficients

  • Eliminate compounds with poor dose-response relationships

• Mechanism of action studies:

  • Determine inhibition mode (competitive, uncompetitive, noncompetitive)

  • Measure Ki values with respect to GMP and ATP

  • Evaluate time-dependence of inhibition

• Selectivity assessment:

  • Test inhibitors against human gmk and gmk from related bacteria

  • Calculate selectivity indices

  • Prioritize compounds with high selectivity for P. amoebophila gmk

• Structure-activity relationship analysis:

  • Group inhibitors by chemical scaffold

  • Identify pharmacophore features correlated with activity

  • Guide synthetic optimization of promising leads

• Advanced validation methods:

  • Thermal shift assays to confirm direct binding

  • Surface plasmon resonance to measure binding kinetics

  • Crystallography of enzyme-inhibitor complexes (if feasible)

This systematic approach enables identification of potent, selective inhibitors with well-characterized mechanisms of action, which could serve as chemical probes for studying P. amoebophila metabolism or as starting points for antimicrobial development.

What strategies can overcome stability issues when working with recombinant P. amoebophila gmk?

Addressing stability challenges with recombinant P. amoebophila gmk requires a multi-faceted approach:

• Expression optimization:

  • Lower induction temperature (16-20°C) to slow folding and prevent aggregation

  • Reduce IPTG concentration (0.1-0.2 mM) to decrease expression rate

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J) to assist folding

  • Use specialized E. coli strains (Arctic Express, SHuffle) designed for difficult proteins

• Buffer optimization:

  • Screen multiple buffer systems (HEPES, phosphate, Tris) at different pH values (6.5-8.5)

  • Include stabilizing additives:

    • Glycerol (10-20%) to prevent aggregation

    • DTT or β-mercaptoethanol (1-5 mM) to maintain reduced state

    • NaCl or KCl (50-300 mM) to shield surface charges

    • MgCl₂ (1-5 mM) to stabilize nucleotide binding site

• Storage conditions:

  • Test protein stability at different temperatures (4°C, -20°C, -80°C)

  • Evaluate cryoprotectants (glycerol, sucrose) for freeze-thaw stability

  • Determine if flash-freezing in liquid nitrogen preserves activity better than slow freezing

  • Consider lyophilization with appropriate excipients for long-term storage

• Fusion partners and solubility tags:

  • Test expression with different tags:

    • Thioredoxin (TrxA) or glutathione S-transferase (GST) for solubility enhancement

    • SUMO or MBP for improved folding and solubility

    • Consider tag position (N-terminal vs. C-terminal) effects on folding

• Enzyme stabilization techniques:

  • Add substrate analogs or product during purification to stabilize active conformation

  • Test chemical cross-linking for stabilizing quaternary structure

  • Consider consensus engineering to improve intrinsic stability

Systematic application of these approaches can significantly improve the stability and handling properties of recombinant P. amoebophila gmk.

How can researchers accurately assess the impact of P. amoebophila gmk inhibition in biological systems?

Accurately assessing the biological impact of P. amoebophila gmk inhibition requires specialized approaches due to the organism's obligate intracellular lifestyle:

• In vitro enzyme inhibition studies:

  • Establish dose-response relationships for inhibitors

  • Determine inhibition mechanism and kinetic parameters

  • Compare effects on P. amoebophila gmk versus host (Acanthamoeba) gmk

• Cell culture models:

  • Acanthamoeba infected with P. amoebophila provides the most relevant model

  • Establish methods to quantify bacterial growth:

    • qPCR targeting P. amoebophila-specific genes

    • Immunofluorescence microscopy to enumerate bacterial inclusions

    • Transmission electron microscopy for detailed morphological assessment

• Metabolic impact assessment:

  • Measure nucleotide pools using LC-MS/MS after inhibitor treatment

  • Monitor GTP/GDP/GMP levels to confirm on-target effects

  • Assess global metabolic changes to identify compensatory mechanisms

• Genetic approaches (if feasible):

  • Attempt conditional knockdown of gmk expression in P. amoebophila

  • Create point mutations in gmk to generate inhibitor-resistant variants

  • Express heterologous gmk to test for functional complementation

• Host-pathogen interaction analysis:

  • Evaluate effects on bacterial developmental cycle

  • Assess changes in host cell responses

  • Monitor bacterial stress responses (e.g., (p)ppGpp accumulation)

• Data integration:

  • Correlate enzymatic inhibition with bacterial growth inhibition

  • Establish pharmacokinetic/pharmacodynamic relationships

  • Build mathematical models to predict effective inhibition parameters

These approaches collectively provide robust evidence for the biological consequences of gmk inhibition and help validate gmk as a potential therapeutic target.

How does the study of P. amoebophila gmk contribute to understanding the evolution of host-pathogen interactions?

Studying P. amoebophila gmk provides unique insights into the evolution of host-pathogen interactions through several perspectives:

• Metabolic dependency relationships:

  • P. amoebophila's retention of gmk while losing de novo nucleotide synthesis pathways illustrates a fundamental aspect of host dependency

  • This exemplifies how intracellular organisms evolve to exploit host resources through targeted enzyme retention

• Evolutionary adaptation signatures:

  • Comparison of P. amoebophila gmk with gmk from pathogenic Chlamydiaceae reveals adaptations to different host environments (amoeba vs. mammalian cells)

  • Sequence changes in substrate binding regions may reflect adaptation to different intracellular nucleotide concentrations or compositions

• Regulatory network evolution:

  • Changes in gmk regulation (e.g., (p)ppGpp sensitivity) between environmental Chlamydiae and pathogenic species illuminate the evolution of stress responses

  • Transcriptional regulation patterns during the developmental cycle provide insights into adaptation to complex host environments

• Reductive evolution principles:

  • P. amoebophila represents an intermediate state between free-living bacteria and highly reduced obligate pathogens

  • The conservation of gmk across this evolutionary spectrum highlights its essential nature

  • The functional analysis of gmk helps define the minimal metabolic requirements for intracellular survival

• Horizontal gene transfer assessment:

  • Unusual features in P. amoebophila gmk sequence or structure might indicate horizontal gene transfer events

  • Such events could represent pivotal moments in the evolution of host adaptation

• Ancient symbiosis model:

  • P. amoebophila's relationship with amoebae represents an ancient host-symbiont relationship

  • Understanding how gmk functions in this system provides insights into the early evolution of intracellular lifestyles

This research contributes to our fundamental understanding of how metabolic dependencies evolve during the transition to an intracellular lifestyle, with broader implications for host-pathogen co-evolution.

What computational approaches are most effective for structural prediction and virtual screening of P. amoebophila gmk?

For effective structural prediction and virtual screening of P. amoebophila gmk, several computational approaches can be leveraged:

• Homology modeling:

  • Identify suitable templates from structurally characterized bacterial gmk enzymes (>30% sequence identity preferred)

  • Use multiple templates for challenging regions

  • Employ advanced modeling tools such as:

    • AlphaFold2 or RoseTTAFold for enhanced accuracy

    • MODELLER for template-based modeling with refinement

    • SWISS-MODEL for automated modeling

• Model refinement and validation:

  • Refine using molecular dynamics simulations (50-100 ns)

  • Validate using:

    • PROCHECK for stereochemical quality

    • VERIFY3D for structural compatibility with sequence

    • QMEANDisco for distance-dependent model quality estimation

• Active site analysis:

  • Identify conserved catalytic residues through multiple sequence alignment

  • Characterize binding pocket properties:

    • Volume and shape using CASTp or POCASA

    • Electrostatic properties using APBS

    • Hydrophobicity distribution

• Virtual screening workflow:

  • Prepare diverse compound libraries:

    • Known nucleotide analogs and kinase inhibitors

    • Natural product databases

    • Fragment libraries

  • Implement hierarchical screening:

    • Pharmacophore filtering based on key interactions

    • Molecular docking using Glide, AutoDock Vina, or GOLD

    • MM-GBSA rescoring for improved ranking

• Advanced simulation techniques:

  • Molecular dynamics for binding mode stability assessment

  • Free energy calculations (MM-PBSA, FEP) for binding affinity estimation

  • Residence time prediction for promising inhibitors

• Machine learning integration:

  • Develop predictive models for activity using known kinase inhibitor data

  • Implement deep learning methods for binding affinity prediction

  • Use active learning to guide subsequent experimental testing

• Visualization and interpretation:

  • Analyze protein-ligand interactions using:

    • Hydrogen bond networks

    • Hydrophobic contacts

    • π-stacking interactions

  • Compare binding modes across multiple compounds to define structure-activity relationships

These computational approaches provide a comprehensive framework for structure-based inhibitor discovery targeting P. amoebophila gmk, accelerating the identification of potent and selective compounds.