Recombinant Acinetobacter sp. Phosphoglycerate kinase (pgk)

What is phosphoglycerate kinase (PGK) in Acinetobacter species?

Phosphoglycerate kinase in Acinetobacter baumannii (AbPGK) is a critical enzyme involved in the glycolytic pathway, catalyzing the reversible conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate with concurrent ATP generation. The enzyme has been identified as a potential antibiotic development target due to its essential role in bacterial energy metabolism . The AbPGK is a 41,260 Da protein that functions in the key energy-producing glycolysis pathway, making it vital for bacterial survival and pathogenicity .

How does Acinetobacter PGK differ structurally from other bacterial PGKs?

Acinetobacter baumannii PGK crystallizes in space group P2221 with unit-cell parameters a = 73.73, b = 177.85, c = 237.49 Å, differentiating it from other bacterial PGKs . Crystallographic analysis reveals that AbPGK can have up to eight molecules per asymmetric unit with a calculated Matthews coefficient of 2.36 ų Da⁻¹ and a solvent content of 47.85% . While sharing some structural similarities with E. coli PGK (which is used as a search model for molecular replacement), AbPGK exhibits distinct structural characteristics that reflect its adaptation to Acinetobacter's metabolic requirements.

What are the optimal conditions for AbPGK activity?

While specific activity conditions for Acinetobacter PGK aren't explicitly detailed in the available data, inference from related phosphate-metabolizing enzymes in Acinetobacter suggests several key parameters. For comparison, Acinetobacter polyphosphate kinase (PPK) shows optimal activity at pH 7-8, significantly reduced activity at lower pH values, and maximum activity at 40°C . Additionally, magnesium concentration plays a crucial role, with optimal activity for similar kinases occurring at approximately 3 mM MgCl₂ . These parameters likely serve as a starting point for optimization of AbPGK activity assays.

What expression systems are most effective for recombinant Acinetobacter PGK?

The most effective expression system documented for AbPGK utilizes E. coli with Gateway cloning vectors . Specifically, the AbPGK gene from Acinetobacter baumannii strain ATCC19606 can be amplified using PCR with primers containing ends that enable incorporation into Gateway cloning vectors . The expression protocol involves:

• PCR amplification of the PGK gene (gi:260555211) from A. baumannii strain ATCC19606

• Gel purification of the PCR product (approximately 1288 base pairs)

• Insertion into plasmid pDONR221 via homologous recombination

• Subsequent cloning into the expression vector pET-57 DEST

• Expression with amino-terminal hexahistidine and NusA tags to enhance target protein solubilization and purification

This system provides reliable expression of functional recombinant AbPGK suitable for structural and biochemical studies.

What purification strategies yield the highest purity AbPGK?

The purification strategy yielding homogeneous AbPGK involves affinity chromatography using the hexahistidine tag, followed by tag removal and additional purification steps . The purification protocol includes:

• Initial capture using Ni-affinity chromatography targeting the hexahistidine tag

• Tag removal using recombinant TEV protease, leaving a single glycine residue at the amino-terminus

• Separation from cleaved tags and uncleaved protein through a second Ni-affinity chromatography step

• Final polishing using size-exclusion chromatography if needed

This multi-step approach has been demonstrated to produce AbPGK purified to homogeneity suitable for crystallization and enzymatic studies .

What crystallization conditions are optimal for Acinetobacter PGK?

The optimal crystallization conditions for AbPGK involve using lithium sulfate as the precipitant . The detailed crystallization protocol includes:

• Preparation of purified AbPGK at a concentration of 10-20 mg/ml in a suitable buffer

• Setting up crystallization trials using the hanging-drop vapor-diffusion method

• Using lithium sulfate as the primary precipitant

• Incubation at 20°C until crystal formation (typically several days)

• Crystal harvesting and cryoprotection prior to diffraction analysis

Under these conditions, AbPGK forms crystals belonging to space group P2221 that diffract to a resolution of 2.5 Å using synchrotron radiation .

How is X-ray crystallographic data for AbPGK typically collected and processed?

X-ray crystallographic data collection for AbPGK involves synchrotron radiation, with detailed parameters as follows:

Data processing typically involves molecular replacement using E. coli PGK (PDB entry 1zmr) as the search model .

How does genomic recombination affect the pgk gene in Acinetobacter populations?

Genomic recombination plays a significant role in Acinetobacter evolution, potentially affecting metabolic genes like pgk. Studies have shown that homologous recombination can occur across approximately 20% of Acinetobacter genomes, contributing to strain diversification . This recombination frequently involves genes encoding proteins exposed to the cell surface or those synthesizing cell-surface molecules . While pgk is not explicitly mentioned among these recombinant regions, the widespread nature of recombination in Acinetobacter genomes suggests that metabolic genes could be subject to similar evolutionary pressures, particularly when advantageous for adaptation to new environments.

What genetic tools are available for studying pgk in Acinetobacter species?

Several advanced genetic tools are available for manipulating and studying pgk and other genes in Acinetobacter species:

• CRISPR-Cas based approaches: A modified CRISPR-Cas9 system has been developed specifically for Acinetobacter, featuring exogenous recombination systems to enhance homologous recombination efficiency . This system uses a two-plasmid approach with RecAb from A. baumannii IS-123 strain, which provides >10-fold higher efficiency than recombinases from other species .

• Splice overlap extension (SOE) PCR and allelic exchange: This technique can generate markerless gene deletions through a two-step process: first introducing a kanamycin cassette flanked by FRT sites and regions matching the target gene, then expressing Flp recombinase to excise the cassette .

• Gateway cloning systems: As demonstrated with AbPGK, Gateway technology facilitates efficient cloning and expression of Acinetobacter genes in heterologous hosts .

• Complementation systems: Plasmids like pBASE can be used for complementation studies, allowing the reintroduction of functional genes with their native ribosome binding sites .

These tools provide researchers with versatile options for genetic manipulation of pgk in both laboratory strains and clinical isolates of Acinetobacter.

How should kinetic assays be designed for accurate measurement of Acinetobacter PGK activity?

Designing robust kinetic assays for AbPGK requires careful consideration of several factors:

• Buffer composition: Based on related kinases in Acinetobacter, a buffer system maintaining pH between 7.0-8.0 is recommended, as significant reductions in activity occur at lower pH values .

• Magnesium concentration: Precise control of Mg²⁺ concentration is critical, with approximately 3 mM MgCl₂ suggested as a starting point based on optimal conditions for related kinases .

• Temperature control: Maintain assay temperature at 37-40°C for optimal activity measurement, as maximum activity for similar Acinetobacter kinases occurs at approximately 40°C .

• Substrate concentrations: Determine the Km for ATP (for comparison, the half-saturation ATP concentration for Acinetobacter PPK is approximately 1 mM) . Use substrate concentrations spanning 0.2-5× Km to accurately determine kinetic parameters.

• Coupled enzyme assays: Consider using NAD⁺/NADH-coupled assays where the production or consumption of ATP is linked to measurable changes in NADH concentration, which can be monitored spectrophotometrically at 340 nm.

• Controls: Include enzyme-free and substrate-free controls to account for background reactions.

How can isothermal titration calorimetry be used to study AbPGK-substrate interactions?

Isothermal titration calorimetry (ITC) provides valuable thermodynamic information about AbPGK-substrate interactions:

• Sample preparation: Purify AbPGK to >95% homogeneity and dialyze extensively against the experimental buffer to minimize buffer mismatch effects. Prepare substrate solutions in the identical buffer.

• Experimental parameters:

  • Protein concentration: 10-50 μM in the cell

  • Ligand concentration: 10-20× higher than protein concentration in the syringe

  • Temperature: Typically 25°C, but can be varied to determine temperature-dependent parameters

  • Reference power: 10-15 μcal/sec

  • Injection parameters: 15-20 injections of 2-3 μL each, with 180-300 second spacing

• Data analysis: Fit the resulting binding isotherms to appropriate models (typically one-site binding) to determine:

  • Binding stoichiometry (n)

  • Association constant (Ka)

  • Enthalpy change (ΔH)

  • Entropy change (ΔS)

  • Gibbs free energy change (ΔG)

• Competitive binding: For multi-substrate enzymes like PGK, perform competitive binding experiments to understand substrate binding order and cooperativity.

This approach provides a complete thermodynamic profile of AbPGK-substrate interactions, complementing kinetic studies and providing insight into the enzyme's catalytic mechanism.

Why is Acinetobacter PGK considered a potential antibiotic target?

Acinetobacter baumannii PGK is considered a promising antibiotic target for several key reasons:

• Essential metabolic role: PGK catalyzes a critical step in glycolysis, an essential energy-producing pathway for bacterial survival . Inhibition of this enzyme would significantly impair bacterial metabolism.

• Increasing drug resistance: A. baumannii has been classified as a top priority pathogen by the World Health Organization due to its widespread resistance to multiple classes of antibiotics . The "enormous increase in multidrug resistance among hospital isolates and the recent emergence of pan-drug-resistant strains" necessitates new antibiotic targets .

• Structural information availability: The successful crystallization and structural characterization of AbPGK provides crucial information for structure-based drug design approaches .

• Potential selectivity: While PGK is conserved across species, structural differences between bacterial and human PGK could potentially be exploited to develop selective inhibitors with minimal host toxicity.

• Nosocomial infection relevance: As A. baumannii is "a significant cause of nosocomial infections among hospital patients worldwide" , targeting PGK could help address a major public health concern.

What approaches are recommended for screening potential AbPGK inhibitors?

A comprehensive approach to screening potential AbPGK inhibitors should include:

• High-throughput enzymatic assays:

  • Develop a spectrophotometric or fluorescence-based coupled assay suitable for 96 or 384-well format

  • Optimize for Z' factor >0.7 to ensure assay robustness

  • Screen compound libraries at concentrations of 1-10 μM initially

  • Include appropriate positive controls (known enzyme inhibitors) and negative controls (vehicle only)

• Structure-based virtual screening:

  • Utilize the solved crystal structure of AbPGK (resolution 2.5 Å) for in silico docking studies

  • Focus on the active site and substrate binding pockets

  • Prioritize compounds with favorable predicted binding energies and that form key interactions with catalytic residues

• Fragment-based screening:

  • Use thermal shift assays (differential scanning fluorimetry) to identify fragments that bind to AbPGK

  • Employ NMR-based methods like saturation transfer difference (STD) to confirm binding

  • Link or grow promising fragments to develop more potent inhibitors

• Whole-cell validation:

  • Test promising compounds for growth inhibition of A. baumannii

  • Compare effects on wild-type and PGK-overexpressing strains to confirm on-target activity

  • Evaluate activity against drug-resistant clinical isolates

• Selectivity profiling:

  • Test activity against human PGK to identify compounds with selectivity for the bacterial enzyme

  • Profile against other glycolytic enzymes to assess pathway specificity

This multi-faceted approach maximizes the chances of identifying selective AbPGK inhibitors with potential for development into novel antibiotics targeting drug-resistant A. baumannii.

How can researchers address the challenge of working with multi-drug resistant Acinetobacter strains in PGK studies?

Working with multi-drug resistant (MDR) Acinetobacter strains presents significant challenges for genetic manipulation studies. Researchers can employ several specialized approaches:

• Alternative selection markers:

  • Use non-clinical antibiotic markers to avoid interference with resistance profiles

  • Employ non-antibiotic selection systems such as tellurite resistance

• CRISPR-based genome editing optimized for clinical isolates:

  • Utilize the modified two-plasmid CRISPR-Cas9 system with RecAb from A. baumannii

  • This system provides >10-fold higher efficiency in clinical isolates compared to standard approaches

• Markerless deletion strategies:

  • Implement FRT/Flp recombinase systems for generating scarless gene deletions

  • Use counter-selection markers like sacB (conferring sucrose sensitivity) for selecting loss of plasmid backbone

• Strain-specific optimization:

  • Test transformation conditions specifically optimized for clinical isolates

  • Modify electroporation parameters to overcome potential barriers in MDR strains

  • Consider the use of conjugation-based methods if transformation efficiency is low

• Heterologous expression systems:

  • Express Acinetobacter PGK in surrogate hosts for biochemical and structural studies

  • Use Gateway cloning vectors as demonstrated for AbPGK

These approaches enable researchers to overcome the challenges associated with genetic manipulation of MDR Acinetobacter strains, facilitating comprehensive studies of PGK and other potential drug targets in clinically relevant isolates.