Recombinant Acinetobacter sp. Cytidylate kinase (cmk)

What is the molecular weight and structural characteristics of recombinant Acinetobacter sp. cmk?

Based on homologous proteins from related bacteria, recombinant Acinetobacter sp. cmk typically has a molecular weight of approximately 25-28 kDa. The enzyme belongs to the P-loop containing nucleoside triphosphate hydrolase superfamily, featuring the characteristic Walker A motif (P-loop) for ATP binding.

While the specific crystal structure of Acinetobacter sp. cmk has not been fully characterized in the provided search results, the enzyme likely shares structural similarity with other bacterial cytidylate kinases, featuring:

• A core P-loop NTPase domain

• CMP binding site with specificity-determining residues

• ATP/GTP binding pocket

• Conformational changes upon substrate binding, particularly involving a "lid" region that stabilizes upon CMP binding

How does Acinetobacter sp. cmk differ from cmk in other bacterial species?

Acinetobacter sp. cmk shares functional similarity with other bacterial cytidylate kinases but may possess unique characteristics related to substrate specificity and regulation. Key differences include:

• Cofactor specificity: While cmk enzymes from some bacteria like E. coli can use either GTP or ATP as phosphate donors, others like Bacillus subtilis cmk cannot use GTP . The specific cofactor preference of Acinetobacter sp. cmk merits further investigation.

• Sequence conservation: Acinetobacter cmk likely shares moderate sequence homology (approximately 45-50%) with other bacterial homologs, similar to the homology observed between other bacterial cmk enzymes .

• Domain architecture: Unlike Bifidobacterium, which possesses a fusion protein with cmk fused to an EngA domain (BiCMKEngA), Acinetobacter species typically have a standalone cmk enzyme .

• Conformational dynamics: Like other cmk enzymes, Acinetobacter cmk likely undergoes substrate-assisted conformational changes upon CMP binding, which are essential for subsequent ATP/GTP binding .

What are the optimal expression systems for producing recombinant Acinetobacter sp. cmk?

The expression of recombinant Acinetobacter sp. cmk can be achieved using several bacterial expression systems, with E. coli being the most common choice. Based on methodologies for similar enzymes, the following systems are recommended:

• E. coli BL21(DE3): This strain is preferred due to its deficiency in lon and ompT proteases, which reduces protein degradation. Common expression vectors include pET series vectors (particularly pET28a with an N-terminal His-tag) under the control of the T7 promoter.

• Expression conditions:

  • Induction with 0.5-1 mM IPTG at OD600 of 0.6-0.8

  • Post-induction growth at 25-30°C for 4-6 hours (rather than 37°C) to enhance soluble protein production

  • Use of LB or 2xYT media supplemented with appropriate antibiotics

• Alternative expression hosts: For proteins that form inclusion bodies in E. coli, alternative hosts like E. coli Arctic Express or Rosetta strains can be considered to enhance proper folding.

While not specifically mentioned for Acinetobacter cmk, fusion partners such as GST, MBP, or SUMO can improve solubility if the native protein shows poor expression.

What is the recommended purification protocol for recombinant Acinetobacter sp. cmk?

A multi-step purification approach is recommended for obtaining high-purity recombinant Acinetobacter sp. cmk:

• Cell lysis:

  • Resuspend cell pellet in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 5 mM β-mercaptoethanol)

  • Lyse cells by sonication or high-pressure homogenization

  • Clarify lysate by centrifugation at 15,000-20,000 × g for 30 minutes at 4°C

• Immobilized Metal Affinity Chromatography (IMAC):

  • Apply clarified lysate to Ni-NTA or cobalt resin

  • Wash with increasing imidazole concentrations (20-50 mM)

  • Elute with high imidazole (250-300 mM)

• Size Exclusion Chromatography (SEC):

  • Further purify using Superdex 75 or Superdex 200 column

  • Typical buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT

• Optional tag removal:

  • If a cleavable tag was used, treat with appropriate protease (TEV, thrombin, etc.)

  • Perform a second IMAC step to remove the cleaved tag

• Quality control:

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

  • Verify identity by western blot or mass spectrometry

  • Check activity using enzymatic assays

The purified protein should be stored in a stabilizing buffer (typically 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT) at -80°C for long-term storage.

How can one assess the activity and quality of purified recombinant Acinetobacter sp. cmk?

Several methods can be employed to assess the activity and quality of purified recombinant Acinetobacter sp. cmk:

• Enzyme activity assays:

  • NADH-coupled spectrophotometric assay: This couples ADP formation to NADH oxidation through pyruvate kinase and lactate dehydrogenase. A typical reaction mixture contains 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM KCl, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 0.5 mM ATP, 2 units each of lactate dehydrogenase and pyruvate kinase, and varying concentrations of CMP/dCMP (0.05-0.8 mM). The decrease in absorbance is monitored at 340 nm .

  • Direct ADP formation assay: Using methods like malachite green to detect released phosphate or HPLC to quantify nucleotide conversion.

• Protein quality assessment:

  • Thermal shift assay (DSF): To evaluate protein stability and proper folding

  • Dynamic light scattering (DLS): To assess homogeneity and detect aggregation

  • Circular dichroism (CD): To confirm secondary structure elements

• Kinetic parameter determination:

  • Determine Km values for different substrates (CMP, dCMP) and cofactors (ATP, GTP)

  • Calculate Vmax and kcat to assess catalytic efficiency

  • Develop Michaelis-Menten plots to visualize enzyme kinetics

Table 1: Typical kinetic parameters for bacterial cytidylate kinases

Note: These parameters are based on typical values for bacterial cmk enzymes and should be experimentally determined for Acinetobacter sp. cmk.

What are the kinetic properties of Acinetobacter sp. cmk with various substrates?

Acinetobacter sp. cmk likely exhibits distinct kinetic properties with various substrates and cofactors. Although specific kinetic data for Acinetobacter cmk is not directly provided in the search results, enzymatic characterization can be performed using standard assays:

• Substrate specificity: While primarily phosphorylating CMP and dCMP, testing against other nucleoside monophosphates (UMP, AMP, GMP) would reveal any promiscuity.

• Phosphate donor preference: Like other bacterial cmk enzymes, Acinetobacter cmk may show preference between ATP and GTP as phosphate donors. Some bacterial cmk enzymes like those from E. coli can use either GTP or ATP, while others like B. subtilis cmk cannot use GTP .

• Reaction conditions optimization:

  • pH optimum (typically 7.0-8.0)

  • Temperature optimum (likely 30-37°C, corresponding to Acinetobacter growth conditions)

  • Metal ion dependency (Mg²⁺ is typically required, but Mn²⁺ may also support activity)

  • Ionic strength effects (KCl concentration optimization)

• Inhibition studies: Testing product inhibition (by CDP/dCDP) and feedback regulation mechanisms would provide insights into metabolic regulation.

The enzyme kinetics can be determined using the NADH-coupled reduction assay as described for BiCMKEngA, with careful consideration that this assay cannot be used for Km determination of ATP/GTP due to the allosteric inhibitory effect of ATP on pyruvate kinase .

How does Acinetobacter sp. cmk function in nucleotide metabolism pathways?

Cytidylate kinase occupies a central position in pyrimidine nucleotide metabolism in Acinetobacter species:

• Primary metabolic role: cmk catalyzes the phosphorylation of CMP to CDP and dCMP to dCDP, providing essential precursors for RNA and DNA synthesis, respectively .

• Pathway integration:

  • Pyrimidine salvage pathway: Recycles CMP from RNA degradation

  • De novo pyrimidine synthesis: Connects with the CTP synthase reaction

  • DNA precursor formation: Provides dCDP that is further phosphorylated to dCTP

• Metabolic regulation:

  • Likely subject to feedback inhibition by downstream products

  • May be regulated at the transcriptional level in response to cellular nucleotide pools

  • Potentially coordinated with other enzymes in nucleotide metabolism

• Relationship to bacterial growth:

  • Essential for cell division due to its role in nucleic acid precursor synthesis

  • Potential connection to ribosome biogenesis through nucleotide supply

  • Activity may vary during different growth phases

What structural features determine substrate specificity in Acinetobacter sp. cmk?

Although the specific crystal structure of Acinetobacter sp. cmk is not detailed in the search results, general structural features that determine substrate specificity in bacterial cytidylate kinases can be inferred:

• CMP/dCMP binding site:

  • A specific binding pocket that recognizes the cytosine base through hydrogen bonding and π-stacking interactions

  • Residues that interact with the 2'-hydroxyl group of the ribose, determining discrimination between CMP and dCMP

  • Coordination of the 5'-phosphate group through basic residues

• Nucleotide-binding pocket features:

  • P-loop motif (Walker A) for binding ATP/GTP phosphate groups

  • Conserved residues for Mg²⁺ coordination, essential for catalysis

  • Specificity-determining residues that may confer preference for ATP versus GTP

• Conformational changes:

  • A "lid" region that likely becomes stabilized upon CMP binding, as observed in the cmk homologue from Thermatoga maritima

  • Substrate-induced conformational changes that create the optimal active site geometry

  • Domain movements that bring the phosphate donor and acceptor into proper orientation

• Catalytic residues:

  • Conserved residues that facilitate phosphoryl transfer

  • Amino acids involved in transition state stabilization

  • Residues that may contribute to the release of products

Modeling studies, as mentioned for BiCMKEngA, can help delineate the specificity governing factors for ligands (dCMP, CMP) and cofactors (phosphate donors - ATP, GTP) in Acinetobacter sp. cmk .

How can recombinant Acinetobacter sp. cmk be used in antibiotic resistance studies?

Recombinant Acinetobacter sp. cmk offers several valuable applications in antibiotic resistance research:

• Target-based drug discovery:

  • As an essential enzyme in nucleotide metabolism, cmk represents a potential antibiotic target

  • Similar to EngA, which was shown to be a potential drug target against tuberculosis and other bacterial diseases

  • High-throughput screening assays using recombinant cmk can identify inhibitors specific to Acinetobacter sp.

• Comparative studies between resistant and susceptible strains:

  • Investigating potential mutations or expression differences in cmk between multidrug-resistant (MDR) and susceptible Acinetobacter strains

  • Structure-function analyses to determine if cmk variants contribute to fitness in antibiotic stress conditions

• Metabolic adaptation mechanisms:

  • Studying how nucleotide metabolism enzymes like cmk adapt in response to antibiotic pressure

  • Understanding if changes in cmk activity correlate with resistance mechanisms

• Species-specific inhibitor design:

  • Exploiting structural differences between human and Acinetobacter cmk for selective targeting

  • Development of species-specific antibiotics, as suggested for designing highly specific and potent antibiotics against pathogens based on variations in essential enzymes

Given that approximately 50% of Acinetobacter baumannii isolates demonstrate multi-drug resistance through various mechanisms , studying essential metabolic enzymes like cmk provides an alternative approach to combating resistance.

What advanced structural biology techniques are most suitable for studying Acinetobacter sp. cmk?

Several advanced structural biology techniques are particularly valuable for studying Acinetobacter sp. cmk:

• X-ray crystallography:

  • Most definitive method to determine atomic-level structure

  • Requires crystallization optimization for Acinetobacter cmk

  • Co-crystallization with substrates (CMP/dCMP) and cofactors (ATP/GTP) to capture different functional states

  • Resolution of 2.0 Å or better would reveal detailed active site architecture

• Cryo-electron microscopy (Cryo-EM):

  • Useful for capturing different conformational states without crystallization

  • Particularly valuable if cmk forms larger complexes with other proteins

  • Single-particle analysis to determine structures at near-atomic resolution

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Ideal for studying dynamics and ligand binding in solution

  • ¹H-¹⁵N HSQC experiments to monitor conformational changes upon substrate binding

  • Chemical shift perturbation analysis to map binding interfaces

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Provides insights into protein dynamics and conformational changes

  • Particularly useful for mapping regions that undergo structural changes upon substrate binding

  • Can reveal allosteric networks within the protein structure

• Molecular dynamics (MD) simulations:

  • Computational approach to model protein dynamics and substrate interactions

  • Can predict conformational changes and identify potential allosteric sites

  • Useful for hypothesis generation before experimental validation

The combination of these techniques would provide comprehensive structural insights into substrate recognition, conformational changes, and the catalytic mechanism of Acinetobacter sp. cmk.

What are the challenges in expressing and characterizing Acinetobacter sp. cmk and how can they be overcome?

Researchers working with recombinant Acinetobacter sp. cmk may encounter several challenges:

• Protein solubility issues:

  • Challenge: Recombinant expression may result in inclusion bodies

  • Solution: Optimize expression conditions by lowering temperature (16-25°C), reducing inducer concentration, using solubility-enhancing fusion tags (MBP, SUMO), or employing specialized E. coli strains (Arctic Express, Rosetta)

• Protein stability concerns:

  • Challenge: Purified enzyme may show limited stability

  • Solution: Screen different buffer conditions with thermal shift assays; add stabilizing agents (glycerol, reducing agents); identify optimal pH and salt concentrations; consider additives like nucleotides that may stabilize specific conformations

• Enzymatic activity measurement challenges:

  • Challenge: The NADH-coupled assay cannot be used for Km determination of ATP/GTP due to allosteric inhibition of pyruvate kinase by ATP

  • Solution: Develop alternative assays such as direct ADP detection methods, radiometric assays with γ-³²P-ATP, or HPLC-based nucleotide quantification

• Structural heterogeneity:

  • Challenge: Multiple conformational states may complicate structural studies

  • Solution: Use substrate/product analogs to trap specific conformations; perform limited proteolysis to identify stable domains; employ crosslinking approaches to stabilize specific states

• Species-specific characterization:

  • Challenge: Limited information specifically on Acinetobacter cmk

  • Solution: Perform comparative studies with well-characterized cmk enzymes from other bacteria; leverage genomic and structural prediction tools to identify unique features

Table 2: Troubleshooting strategies for recombinant Acinetobacter sp. cmk expression and characterization

How might Acinetobacter sp. cmk contribute to multidrug resistance mechanisms?

While cmk itself is not directly implicated as a resistance determinant in Acinetobacter species, several hypotheses can be formulated regarding its potential indirect contributions to multidrug resistance:

• Metabolic adaptation: Changes in cmk expression or activity could potentially alter nucleotide pools, affecting DNA repair mechanisms and mutation rates under antibiotic stress.

• Stringent response connection: Like observed in Clostridium cellulovorans under butanol stress , stress conditions in Acinetobacter might trigger upregulation of amino acid biosynthesis and translation machinery components, potentially including nucleotide metabolism enzymes like cmk.

• Energy homeostasis: As an ATP-consuming enzyme, cmk activity might be regulated as part of energy conservation strategies during antibiotic stress.

• Biofilm formation support: Nucleotide metabolism might play roles in supporting biofilm formation, a known resistance mechanism in Acinetobacter baumannii.

• Compensatory mutations: In resistant strains with altered metabolism, compensatory changes in cmk activity might help maintain fitness.

Acinetobacter species have among the largest number and variety of resistance mechanisms of all gram-negative bacilli , and understanding the metabolic adaptations supporting these resistance mechanisms could provide new insights for therapeutic intervention.

What comparative genomic approaches could reveal about cmk evolution in Acinetobacter species?

Comparative genomic approaches would provide valuable insights into cmk evolution across Acinetobacter species:

• Sequence conservation analysis:

  • Evaluate sequence conservation of cmk across different Acinetobacter species

  • Compare with cmk sequences from other bacterial genera to identify Acinetobacter-specific features

  • Determine if clinical isolates, particularly multidrug-resistant strains, show specific cmk sequence variants

• Synteny and genomic context:

  • Analyze the genomic neighborhood of cmk in different Acinetobacter species

  • Determine if cmk is consistently found in operons with other specific genes

  • Investigate if the genomic context differs between environmental and clinical isolates

• Evolutionary rate analysis:

  • Compare synonymous vs. non-synonymous substitution rates to assess selective pressure on cmk

  • Identify regions under stronger purifying selection (likely functional domains)

  • Detect any signatures of adaptive evolution in specific lineages

• Domain architecture comparison:

  • Unlike Bifidobacterium with its CMK-EngA fusion , determine if any Acinetobacter species show alternative domain architectures

  • Assess if any specific strains have cmk gene duplications or variants

• Horizontal gene transfer (HGT) assessment:

  • Evaluate if the cmk gene shows evidence of HGT between Acinetobacter and other bacterial genera

  • Determine if antibiotic-resistant strains show distinct evolutionary histories for cmk

Such analyses would contribute to understanding the evolution of nucleotide metabolism in Acinetobacter and potentially reveal connections to pathogenicity and resistance mechanisms.

How can recombinant Acinetobacter sp. cmk be utilized in developing species-specific inhibitors?

Recombinant Acinetobacter sp. cmk provides an excellent platform for developing species-specific inhibitors through the following approaches:

• Structure-based drug design:

  • Determine high-resolution crystal structures of Acinetobacter cmk in complex with substrates and inhibitors

  • Identify unique structural features that distinguish it from human cytidylate kinases

  • Use computational docking to design compounds that specifically target Acinetobacter cmk

• High-throughput screening approaches:

  • Develop robust assays suitable for screening large compound libraries

  • Implement counterscreens against human cytidylate kinases to ensure selectivity

  • Create fluorescence-based assays for real-time monitoring of inhibitor effects

• Fragment-based drug discovery:

  • Screen libraries of low-molecular-weight fragments for binding to Acinetobacter cmk

  • Determine binding sites using NMR or crystallography

  • Link or grow fragments to develop high-affinity, selective inhibitors

• Allosteric inhibitor development:

  • Identify allosteric sites unique to Acinetobacter cmk

  • Design inhibitors that lock the enzyme in inactive conformations

  • Target protein-protein interaction interfaces if cmk functions in complexes

• Validation in resistant clinical isolates:

  • Test candidate inhibitors against a panel of multidrug-resistant Acinetobacter clinical isolates

  • Evaluate efficacy in combination with existing antibiotics

  • Assess resistance development frequency

This approach aligns with broader strategies to develop species-specific antibiotics by targeting variations in essential enzymes, as mentioned in relation to EngA as a potential drug target against tuberculosis and other bacterial diseases .